Chisa Yukizome, a member of Ultimate Despair later gives a copy of this video to the Future Foundation's founder, Kazuo Tengan, ultimately making him use this brainwashing video as the attacker in the Final Killing Game.
Created by Junko using Ryota's animation, it forced its viewer into a state of trance. While the general thoughts of its victim are unknown, in the case of Makoto Naegi, his trance-state involved him recall the deaths of his former classmates, who had called upon his failure and survivor's guilt, leading him to a state of despair that made him agreeable to suicide as "repentance". Similar guilt-based despair can also be seen from how Ruruka Ando tortured herself before her suicide.
The brainwashing effect, once viewed, cannot be stopped or overpowered; despite Great Gozu stabbing out his own eyes, Seiko Kimura slamming herself repeatedly into the wall, and Ruruka Ando's attempt to use her mind-altering candy on herself, they still ended up killing themselves.
It is also possible, however, that these events were part of their suicides rather than attempts to stop themselves from doing so, as in Ruruka Ando's suicide, her sclera had turned red, possibly due to subconjunctival haemorrhaging due to choking on the vast amount of sweets seen in her mouth.
The brainwashing can only be stopped by inducing unconsciousness, as Makoto was brought out of his brainwashing when Juzo Sakakura forcefully choked him.
Although the exact time of its creation is unknown, the video was first used on the Reserve Course Students as they made their way into, and destroyed Hope's Peak Academy's new building, killing most of the students and staff inside. As a "thank you", Junko emailed all the Reserve Course students Monokuma's Gloomy Sunday. Watching the video forced all the Reserve Course students to commit mass suicide by throwing themselves off the building, and running into the fires. The one exception was Hajime, who was living as Izuru at the time and had aligned himself with Junko, thus making him the only survivor of the Reserve Course mass suicide.
The suicide video would have no known uses until many years later when Chisa handed Kazuo Tengan both Junko's Despair video and the suicide video. Kazuo would go on to use the suicide video in the Final Killing Game, which he had orchestrated himself, another couple years later.
During the events of the Final Killing Game, the closest person to a monitor would be awoken from their drug induced sleep and forced to watch Monokuma's Gloomy Sunday, forcing them to kill themselves. Chisa, Great Gozu, Seiko, and Ruruka would all fall victims to the video and commit suicide. Makoto would also end up watching the video but was stopped from killing himself by Juzo at the last minute.
- The name refers to the song "Gloomy Sunday", also known as "Hungarian Suicide Song" due its depressing nature, and its alleged but unproven relationship between listening and suicide, including the composer's own suicide in 1968. Despite the lack of linkage, the BBC had banned the broadcast of the English vocal version until 2002.
- The video is very similar to the Radical-6 virus from Zero Escape: Virtue's Last Reward (the entire Final Killing Game takes inspiration from the Zero Escape series). The Radical-6 virus is a virus that eventually drives its victim to suicide, much like the Suicide Video.
- Monokuma's Gloomy Sunday is shown in the Danganronpa mutual killing games, building up piece-by-piece every night as the game goes on, sometimes foreshadowing events to come in the game.
- The music played in the background is "Mr. Monokuma's Lesson" but reversed.
- Makoto is the only known survivor of the Suicide Video in the Final Killing Game, thanks to Juzo choking him just before he commited suicide.