Watson's name will be missing from the main draw for only the second time since 2010 after she was beaten 3-6 6-4 7-5 by 16-year-old Czech Sara Bejlek in a rain-affected clash.
Storms in New York meant the clash was interrupted several times and although Watson won the first set, it was her teenage opponent who eventually prevailed.
Twenty-two-year-old Jubb was looking to qualify for a grand slam for the first time. Having come from behind in his first two matches, he again battled back from a set down against Dutchman Gijs Brouwer but fell to a 7-6 (3) 3-6 6-2 loss.
Watson and Jubb's defeats mean there will be seven British players in the main singles draws, which begin on Monday.
Bejlek was the third Czech teenager to win on Friday, joining 17-year-olds Linda Noskova and Linda Fruhvirtova in the main draw, while, on the men's side, former junior champion Wu Yibing became the first Chinese man to qualify for the US Open and was swiftly joined by compatriot Zhang Zhizhen.
Two-time champion Tracy Austin, meanwhile, experienced another emotional moment at Flushing Meadows as she watched her son, Brandon Holt, make it through to the main draw.