A new night watch is hired to look over Nocturnal's Hootery, a pizzeria in Brooklyn. Armed with a flashlight and a SecureTablet 7, the player must survive all five nights in the pizzeria before the animatronic band gets to their office. The backstory is uncovered through various clues and minigames throughout the game. It is revealed Nocturnal's Hootery was once a restaurant named Spot's Funtime Jam and owned by a different company. However, the animatronic Spot tore off someone's limb as shown in a minigame, making the company go bankrupt. The original owners had to sell it to new owners. The new owners had to rename the company Nocturnal's Hootery in order to avoid any negative feedback. The game's events take place in 2015, three years after the new owners rebranded Spot's Funtime Jam. The animatronic's backstories are not seen until Five Nights at Nocturnal's 2: Night Owl.
- Larry Smith - A night watch who's job is to watch the animatronics.
- Victor - A man who gives the player instructions on the phone.
- Phone Girl - An uninterested teenaged girl who's job is to give the player instructions through the phone on Nights 6 and 7.
- Nocturnal the Owl - The main animatronic who wanders through the facility to kill the player. He starts on the Show Stage and is first active on Night 1.
- Iris the Eagle - The opposite of Nocturnal who has her own side-show next to the main stage to the left. She starts on the Left Stage and is first active on Night 1.
- Flyer the Blue Jay - A blue jay animatronic with his own side-show next to the main stage to the right. He starts on the Right Stage and is first active on Night 1.
- Viris the Vixen - A broken-down vixen animatronic who starts in Backstage and is first active on Night 2.
- Backstaged - A spare endoskeleton from the backstage nicknamed "Backstaged" by the employees due to the fact it's never used. He starts in Backstage and is first active on Night 3.
- Spot the Dog - An old, withered animatronic who starts from the backstage area and starts on Night 4.
- Nightmare Nocturnal - The only animatronic in Hootery Night Jam who is a nightmarish version of Nocturnal.
- Robber - A man who appears in the minigames and attempts to break in.