Former British number one Watson, 30, last won direct qualification to a Grand Slam at the 2019 US Open.
And that unwanted record for the world number 161 continued with a 3-6 6-4 7-5 loss to 16-year-old Czech Sara Bejlek.
Jubb, 22, the world number 203, was unable to get past Dutchman Gijs Brouwer, losing 6-7 (3-7) 6-3 2-6.
The final Grand Slam of the year starts on Monday at Flushing Meadows, New York.
Watson’s defeat means there will be two British women in the singles draw, with defending champion Emma Raducanu and Harriet Dart already in as direct entries.
Jubb was seeking to join Andy Murray, Cameron Norrie, Dan Evans, Jack Draper and Kyle Edmund in the men’s draw.