A 90 second video, uploaded to video sharing website YouTube, shows a number of people being hit in the face across in streets and shops across the city centre.
In one scene, a tram driver sitting quietly in his Supertram cab, has a whipped cream pie shoved in his face after one of the pranksters leans through an open window while in another clip, a ginger-haired cyclist is knocked from his bike as he cycles down Mulberry Street after being hit full in the face.
The clip, which has already been viewed more than 5,000 times, also shows a shocked worker at a city branch of sandwich chain Subway reacting in horror after being slammed in the face with a pie while a fairground worker, street cleaner and a tram conductor are also targeted, along with members of the public.
The clip, which is soundtracked by the Benny Hill theme music Yakety Sax, begins showing one of the pranksters, wearing a hoodie, squirting whipped cream into a foil tin.
Entitled "Pie Pranks Sheffield" and uploaded by a user called DankTV, the action then leads into a series of attacks filmed across Sheffield city centre.
In one scene, two pie-wielding men run onto a Supertram and slam a conductor in the face before exiting, laughing.
Further attacks take place on a passenger at a tram stop, on customers inside the city centre branch of McDonalds and also on a street cleaner and members of the public in the Peace Gardens.
Commenting on the video on YouTube, Hasan Ramzan posted: "Too funny" while Areej Khan wrote "Looooool smashed it."
However not all were impressed and one user, johnnyboy78623 wrote: "Not funny but border line assault."
It is not clear when the video was filmed, but the footage was uploaded to YouTube earlier this week.
Custard pies have long been used as a comedic device by clowns in circus performances, in practical jokes for harmless fun and also for throwing into the faces of public figures as a sign of disapproval.
UK Saturday morning programme Tiswas had custard pies as a regular feature and even had a character called The Phantom Flan Flinger, a masked man who pied people.