Djokovic takes on Australian upstart Thanasi Kokkinakis, who had to qualify for the tournament, while Nadal faces world number 120 Andrey Kuznetsov.
Murray, who is in the middle of a 12-match unbeaten run on clay, faces another Australian in Nick Kyrgios, the 29th seed.
In the pick of the rest, men's seventh seed and clay specialist David Ferrer plays Simone Bolelli, ninth seed Marin Cilic plays Leonardo Mayer and exciting youngster Borna Coric meets Jack Sock.
Williams, a two-time winner in the French capital, takes one former world number one Victoria Azarenka, who has dropped down to 27th in the world after struggling with injury over the last 18 months.
Williams holds a 15-3 advantage over the Belarusian, a record which included victories in all her matches on clay against the two-time Australian Open champion.
Also in action on the day is outside favourite and fourth seed Petra Kvitova, the Wimbledon champion.
The southpaw takes one Romania's Irina-Camelia Begu, who she also faced in Madrid earlier this month.
French Open order of play on the show-courts:
Court Philippe Chatrier
Petra Kvitova (CZE x4) v Irina-Camelia Begu (ROM x30)
Novak Djokokic (SRB x1) v Thanasi Kokkinakis (AUS)
Richard Gasquet (FRA x20) v Kevin Anderson (RSA x15)
Serena Williams (USA x1) v Victoria Azarenka (BLR x27)
Court Suzanne Lenglen
Andy Murray (GBR x3) v Nick Kyrgios (AUS x29)
Alison van Uytvanck (BEL) v Kristina Mladenovic (FRA)
Andrey Kuznetsov (RUS) v Rafael Nadal (ESP x6)
Tsvetana Pironkova (BUL) v Sloane Stephens (USA)
Andrea Petkovic (GER x10) v Sara Errani (ITA x17)
David Goffin (BEL v17) v Jeremy Chardy (FRA)
Simone Bolleli (ITA) v David Ferrer (ESP x7)