While the strip began as a gag-based series in which the three main protagonists (Torg, Riff and Zoë) would stumble from one brief, bizarre, parody-centric adventure to the next, the characters and plotlines have gradually become longer and more serious. However, even the more dramatic and soap operatic story arcs often conform to the common gag comic strip format. While there is often sexual innuendo and cartoon violence, the comic contains no strong profanity or explicit nudity.
The characters in Sluggy Freelance are a varied and diverse lot. The primary protagonist Torg is a cheerful and impulsive nerd, who frequently finds himself going on wild adventures (though rarely of his own volition). Often, these adventures are enabled by Torg's genius inventor friend, Riff. Zoë, the most normal character of the bunch, serves as the futile voice of reason for the cast. They are accompanied by their sometimes-witch friend Gwynn, a psychopathic switchblade-wielding rabbit Bun-bun, the hyperactive ferret Kiki, and a shape-shifting alien named Aylee.
Sluggy Freelance has featured several yearly recurring themes, although many of them have eventually been broken or discontinued due to developments in the overall plot.
In an early 1998 plotline, one of Riff's inventions sent Torg to the "Dimension of Pain." Every Halloween afterwards for several years, a different demon was sent to Earth to try to bring him back, failing in amusing and unexpected ways.
Bun-bun has tried to kill Santa Claus every Christmas, with continuously escalating violence; the fact that Bun-bun became the Easter Bunny early on in the strip merely added spice to the relationship. There was a break in the tradition when Bun-bun was thrown out of time and was not present in 2005, and aside from an attack more inconveniencing than dangerous in 2006 he has not resumed the feud.
Also on almost every Christmas/Hanukkah, Torg and Riff have attempted to continue their own, private tradition of giving each other "a beer every year." Usually they never quite get it right, for a variety of reasons, including being trapped in a mummy's tomb, selling their shoulders for science, and other random occurrences.
Every year on the 25th of August, the comic features a small animation to commemorate the comic's anniversary, most of which involve Kiki singing karaoke. The Fifth Anniversary, August 25, 2002, fell on a Sunday, which traditionally was reserved for full color extended comics. This comic combined the two themes, presenting a full-color animated comic, which advanced one frame at a time.
In every New Year's Eve storyline, Bun-bun gets drunk on 151 Rum, which results in his being uncharacteristically kind and courteous (such as apologizing to Torg or praising the main cast).
Abrams invites other well-known webcomic artists to do the strip for a week once or twice a year, while he goes on vacation. A frequent result is a parody of the strip itself, other webcomics, other creative works and/or artists, including Scooby-Doo and Ayn Rand. Clay Yount of Rob and Elliot was guest artist several times prior to taking over Saturday duties.
Baen SF author John Ringo was profoundly affected by Sluggy while writing his Legacy of the Aldenata series; as a result, the crew of a massive mobile artillery platform that first appears in the third book of this series (When the Devil Dances) are depicted as die-hard Sluggy fanatics to comedic effect (up to, and including, naming their vehicle after Bun-Bun and painting a giant picture of Bun-Bun on it). They are joined in the fourth book (Hell's Faire), by a character based on the late friend of Pete Abrams who was the inspiration for Riff. A section of original Sluggy comics set in the alternate future world of the novels appears in the end of Hell's Faire, and a sampler of Sluggy storylines is included on the CD-ROM bound into this book. Pete possibly returned the favor shortly thereafter by entitling one subchapter "Hell's Unfair." Another possible Sluggy reference is in the short story "Lets Go to Prague" where one character uses the codeword Kizke. This is the common mispronunciation of the demon K'z'k. (The proper pronunciation has no vowels.)
Also, the first two novels of Ringo's distant-future Council Wars series have appearances by an irascible, treacherous, switchblade-toting, telemarketer-hating AI in a rabbit-shaped body—created by a long-dead fan of an unnamed 20th-century webcomic.
In S.M. Stirling's Conquistador, one of the characters unleashes a self-destruct with the code word "Override B-1". Override B-1 is a program that causes Sluggy character Oasis to unleash her own level of destruction.
Numerous other webcomics have referenced Sluggy Freelance, and various guest artists on Sluggy Freelance have included their own webcomics' characters in their guest strips, including User Friendly who swapped A.J. for Torg for a week
Additionally, shortly after the birth of Leah Nicole Abrams in the middle of "The Love Potion" storyline, Sluggy Freelance entered a three-week long side story. The story involved Ki and Fooker of General Protection Fault, Lindesfarne and Ralph of Kevin and Kell, and Bruno and Fiona of Bruno the Bandit attempting to play the roles of Sluggy Freelance characters and find the original cast. Other characters, such as Gav from Nukees, and Trudy from General Protection Fault, made appearances. The non-comic characters from Mystery Science Theater 3000 also appear, in their famous silhouetted form.
There are several implicit cross-overs with R.K. Milholland's Something Positive, where Bun-bun is indirectly referred to. Milholland goes as far as imply that Bun-bun was Aubrey Chorde's bunny when she was a teen, and stole her flick-knife when she was forced to give him away by her mother.
In an episode of Freefall, wolf engineer Florence has caught a couple of rabbits for dinner; the robot Helix thinks they are intended as pets and names them Kevin (presumably after Kevin Dewclaw in Kevin and Kell) and Bun-Bun.