The street starts at its junction with the Eastern Outlet, the Brooker Highway and Davey Street, near the historic Royal Engineers Building. It varies between three and four lane for almost all of its length, as it passes through the city. After leaving the CBD, it becomes two-way and extends into the suburb of South Hobart.
Macquarie Street borders city garden Franklin Square. Prominent buildings in the street include the Lands Building, which houses the Department of Primary Industries, Water and Environment, The Mercury building, St Davids Cathedral, and the Federal Reserve Bank building.
Macquarie Street is featured as a property in the Australian version of Monopoly.