American Williams, 40, plays France's Harmony Tan on Centre Court, 12 months on from tearing her hamstring in her first-round match at SW19.
Women's world number one Iga Swiatek opens play on the main show court against Croat Jana Fett from 13:30 BST.
Men's second seed Nadal, a two-time winner, then meets Francisco Cerundolo.
A 23-time Grand Slam singles champion, Williams made her long-awaited return to action in the doubles at Eastbourne last week alongside Ons Jabeur, after ending speculation over her future in the sport by accepting a Wimbledon wildcard.
She is one short of Australian player Margaret Court's record of 24 Grand Slam singles wins.
"I didn't retire. I just needed to heal physically, mentally. I had no plans, to be honest. I just didn't know when I would come back," Williams said.
"I didn't know how I would come back. Obviously Wimbledon is such a great place to be, and it just kind of worked out."
French Open champion Swiatek, 21, enters Wimbledon on an impressive 35-match winning run which has taken her level with Venus Williams for the longest WTA victory streak this century.
The Pole's match against world number 252 Fett is followed by 22-time major winner Nadal's return for the first time since 2019, after the Spanish world number four was able to recover from treatment on a chronic foot injury in time for the Championships.
"I can walk normal almost every single day. That for me is the main issue. When I wake up, I don't have this pain that I was having for the last year and a half, so I'm quite happy about that," said Nadal.
On Court One, Italy's 2021 men's runner-up Matteo Berrettini faces Chilean Cristian Garin, before 2019 women's champion Simona Halep meets Karolina Muchova.
Men's Greek fourth seed Stefanos Tsitsipas closes play against Swiss player Alexander Ritschard.