NES - Did You Know Gaming? Feat. Caddicarus
Between rounds, Piston Hondo can sometimes be seen reading a manga. The texture used for the pages consists of scanned panels from the Sailor Moon manga series, albeit rearranged and flipped upside down to not look completely identical to the source material.
Contributed by Kitsune Hawk
Each of the opponents were voiced by actors with the same nationalities as them. For example, Christian Bernard (a French Actor) voiced Glass Joe, and Sumit Seru (an Indian Actor) voiced Great Tiger.
Contributed by CuriousUserX90
One of Doc Louis' in-between round quotes while fighting Bald Bull is "Hey, Mac baby! If you can't beat Bald Bull in a fight, how about an arm wrestle?" This is a reference to the 1985 arcade game Arm Wrestling, in which Bald Bull was one of the opponents.
Contributed by Takahashi2212
Several famous Nintendo characters were considered for inclusion in the game, but ultimately dropped to focus on the game's own world and its characters, "rather than simply borrow from existing Nintendo IP."

Princess Peach was considered for an opponent, but ultimately the developers felt that punching a woman out would be inappropriate for the game. Mario did not reprise his role as the referee, or appear as an opponent, because the developers didn't think he would fit well with the game's art style.
Contributed by Laikue
In the World Circuit stadium, Donkey Kong can be spotted in the audience during title defence fights.
Contributed by SOGESNAKE
Looking into the game's files, all of the data on Disco Kid is stored under a file named "kidquick". It's possible that Disco Kid would've originally been Kid Quick from the arcade version of Punch-Out!!.
Contributed by Antwan
The number 13, commonly associated with misfortune, appears as a common (albeit subliminal) motif throughout the fight against Mr. Sandman:
• His full nickname, Mister Sandman, has 13 letters.
• He is the 13th opponent in both Contender and Title Defense modes; reciprocally, Little Mac is Mr. Sandman's 13th opponent, who Sandman loses to.
• Little Mac has 13 hearts by default in both fights.
• Mr. Sandman's introduction in Contender mode has 13 frames, as opposed to the usual 4.
• When Mr. Sandman gets tired after the dreaded Berserker Rage in Title Defense, Little Mac only needs to punch him 13 more times to defeat him (although obviously less punches are required if stars are used).
Contributed by game4brains
Losing 100 matches at any time in the game will unlock "Headgear". This makes it so that hits you receive do less damage. Spoiler:The headgear is also given to Glass Joe during Title Defense Mode, since you gave him his 100th lose.
Contributed by gamemaster1991