Passengers at Heathrow airport near London were already checking in for BA Flight 223 on Friday when the airline announced its cancellation about two hours before it was due to depart at 3:05pm (15:05 GMT).
Thursday's flight was cancelled a few hours before it was scheduled to depart after British Airways received "security advice" from the UK government.
Passengers on the same BA Flight 223 on Wednesday were questioned by FBI agents upon its arrival at Dulles international airport in the US capital.
"Following the latest advice from the UK government received this (Friday) afternoon, British Airways has cancelled today's flight BA 223 from London Heathrow to Washington due to security reasons," the airline said.
"As a consequence of today's cancellation, the return flight BA 222 will also be cancelled (due to depart Washington at 21:40 arriving London Heathrow at 10:00 on Saturday)," it said. The day's third and last flight was still scheduled to depart as normal.
Thursday's cancellation was believed to be the first involving a US-bound British airliner for security reasons since the 11 September attacks in the US in 2001.
Washington and London have been on heightened alert throughout the Christmas and New Year period because of fears of an attack.
Chief among those fears is a repeat of 11 September, when suspected al-Qaida operatives hijacked three US domestic airliners, then flew them into the World Trade Centre in New York and the Pentagon in Washington. A fourth hijacked airliner crashed in Pennsylvania.
Besides the British Airways flights, an Aeromexico flight from Mexico City to Los Angeles was cancelled on New Year's Eve, as well as six Air France flights between Paris and Los Angeles on Christmas Eve.
The British government has recently admitted it was putting armed guards on selected British airline flights to the US.