Australian Open and Wimbledon champion Djokovic closes the day programme at Arthur Ashe Stadium against 89th-ranked Joao Souza of Brazil.
Three-times defending US champion Williams, the Australian, French and Wimbledon champion seeking to become the fourth woman ever to complete a Grand Slam, opens the night programme against 86th-ranked Vitalia Diatchenko of Russia.
The tournament's first match on the Arthur Ashe centre court pits seventh-seeded Ana Ivanovic of Serbia against Dominika Cibulkova of Slovakia, followed by two-times US Open winner Venus Williams against Puerto Rican Monica Puig.
The second Opening Night match under the lights features two-times winner Rafa Nadal of Spain, the eighth seed, against up-and-coming Borna Coric of Croatia, an 18-year-old ranked 35th.