At the end of Daemon Rising, Bob and Dot got engaged. To the confusion of everyone, however, a portal opens from the net and Ray Tracer and another Bob steps through. Inasmuch as the second Bob looks like the original from Seasons 1 and 2, Dot names the Bob which merged with his keytool Glitch Bob. Most of My Two Bobs is taken up with the efforts of Dot, the two Bobs themselves, and the other mainframers to ascertain who is the original and who is the copy and in general to come to terms with the situation.
After some counseling from Phong and Mouse, Dot decides to marry the new Bob, whereupon Glitch Bob -the nominal original- earnestly attempts to return himself to his original form in order to resolve his existential situation. His efforts ultimately fail and leave him in a catatonic, "crystalline" state; he is covered in a starry substance that proves to be impenetrable. Dot continues with her wedding plans as Glitch Bob is treated at the Supercomputer.
Glitch Bob's condition steadily worsens on her wedding day. The impenentrable starry substance covering him gradually dims completely, and the Guardians believe that they have lost him. This moment of crisis prompts all the keytools of other guardians (which had disconnected from guardians infected by Daemon) to separate from Glitch Bob, and they revive him before returning to the guardians. The Guardians discover that his code no longer matches what they have on file, suggesting that he is in fact the copy.
Glitch Bob returns to Mainframe to stop the wedding, where he reveals that Megabyte has previously stolen part of his code. Glitch Bob is able to return this code to Bob, which forces Megabyte to emerge -Megabyte had been disguised as Bob all along!
After a spectacular battle in the church, investigation reveals that Megabyte is now a Trojan Horse virus, able to shapeshift and effectively disguise himself as anyone. Megabyte eludes capture by using various aliases and a doppleganger and ultimately infiltrates the war room by taking on the form of Frisket. After suborning various personnel, including Dot's father, and capturing Enzo, Megabyte gains "complete control" of the Principal Office. The movie ends with him proclaiming that viruses are predatory by nature; his last words, which are the final words of the films, are "Prepare yourselves for the hunt!"
The film includes many parodies of pop culture tropes including, but not limited to, The Brady Bunch, Pokemon, Darth Vader, Star Trek's transporter, 2001: A Space Odyssey, The Village People, The Thing, and The Blues Brothers.