In the Western version of the game, when an enemy is killed, it is possible to cut off their limbs during their death animation. This feature was censored in the Japanese version of the game to be inline with Japan's rating system.

The description for the Heshikiri Hasabe sword alludes to a real-life event where daimyo Oda Nobunaga used the sword to murder one of his servants in anger after he insulted him. The final enemy in the "Kanbei and the Overlord" side mission is a yōkai resembling a biwa that steals the weapon because of a grudge related to the sword. It is heavily implied that the demon is that slain servant.

During the final stretch of the side mission, "Kanbei and the Overlord", if the player runs to the locked door they can learn the name of the hidden Revenant before the second wave of enemies appears. Its name is Sugitani Zenjubo, a real-life samurai who was captured by daimyo Oda Nobunaga after the Siege of Kanegasaki in 1570 and sentenced to death by beheading. This event is alluded to in the Revenant's dialogue, and in its Blood Grave where it specifically names Nobunaga as the cause of death, the only instance of a Blood Grave with an identified culprit in the game.

The real-life Edward Kelley was an alchemist who worked for the Queen during the time. However, he was arrested by the Roman Empire for failing to create gold and died in prison. The Kelley met in the game is a homunculus created by John Dee months after the original's death. In the second visit to the Tower of London, before the boss, there are clones who have the same memorandum as the first one.

"I am the hollowed man. I am the hollowed man. Many times I've died. Many times rose again."

In the second version of the Tower of London, William can discover a piece of amrita with the voice of a child inside it. This child and the brother he is referring to is a reference to The Princes in the Tower paintings. They are expressions that frequently refer to Prince Edward V (11) and Richard (9), who mysteriously disappeared in the Tower of London in 1483. To this day it was unknown whether the died in the Tower or escaped.

In the post-game version of the Tower of London, there is a man in a prison cell William can talk to. Through the hints he gives about himself, he is likely Walter Raleigh, the man who claims to have found the "City of Gold", El Dorado. This is highlighted by the Large Spirit Stone he gives to William after exhausting his dialogue.

In the Beta version it's shown that the Twilight versions of the bosses were originally going to have minor enemies added to the arena for an extra challenge. This was changed in the final game, where all bosses get an additional unique attack instead.

Throughout the game, there are several NPC Revenants that are references to Team Ninja's works:

Shibasawa Kou - The game's producer
Samurai_Gaiden - Ninja Gaiden
Shadow Warrior - The European name of Ninja Gaiden
RebootRygar - Rygar
GITAROO-MAN - Gitaroo Man
theJaquio - The main villain from Ninja Gaiden
Holy_San - Holy Vigoor Emperor from Ninja Gaiden
Genshin07 - Genshin from Ninja Gaiden II
LOA.test.subject.1 - Lords of Alchemy from Ninja Gaiden 3
IreneIsMyHero - Irene Lew from the original Ninja Gaiden Trilogy
Dr. Smith - Dr. Walter Smith from Ninja Gaiden (NES)
oinin_AYANE - Ayane from Dead or Alive
nukenin_KASUMI - Kasumi from Dead or Alive
ZACK - Zack from Dead or Alive
GARI - Dr. Gari from Tecmo Secret of the Stars
KarmicPunishment - The curse from Ninja Gaiden 3
Hajinmon4Life - Ayane's fighting style, Hajin Mon
dropped_by_izuna - The Izuna Drop move from Ninja Gaiden and Dead or Alive

Nioh's story is based on James Clavell's 1975 novel Shōgun, which focuses on a fictional English samurai who examines the rise of a new Shogunate, based on the real-life William Adams.

The Dark Souls series was a major inspiration for the gameplay, as well as the team's older works, such as Ninja Gaiden and Onimusha. The loot system was also said to be inspired by Diablo.

The development of the game began in 2004 internally by Koei, four years before the merger with Tecmo in 2008. After it was scrapped, production was rebooted and transferred to Omega Force, and shifted the genre to something similar to Dynasty Warriors. This version was also scrapped.

Team Ninja was brought on to help develop the action gameplay in 2010, while earlier staff from the original Koei team handled the scenario, having a finalized concept in 2012. The final version was released in 2017, placing the project in development for between twelve and thirteen years.

The game was originally based on the unfinished Akira Kurosawa film, Oni. Though the idea was dropped in favor of an original story, the artistic elements and battle movements were inspired by other Kurosawa films such as Yojimbo and Seven Samurai.