The absence of the key pair from the session at Carrington casts doubts about their availability for the semi-final second leg against Barca at Old Trafford.
Rooney took a knock to his hip before scoring the equaliser in the 2-1 Premier League defeat at Chelsea on Saturday, while Vidic suffered a facial injury.
United are aiming to book a place in the final in Moscow after a 0-0 draw at the Nou Camp last week.
A four-man group comprising Rio Ferdinand, Owen Hargreaves, Michael Carrick and Wes Brown trained away from the rest of the squad at the beginning of the session.
With United looking to book a place in the final, they will be banking on Rooney to recover on time.
The England striker took his tally for the season to 18 goals at Stamford Bridge.
Serbian centre-back Vidic, who missed the first leg because of a stomach infection, saw just eight minutes of action against Chelsea.
He was stretchered off after taking a knee in the face following a tussle with Didier Drogba.
Darren Fletcher is looking to retain his place in the side after making his comeback from a knee injury against Chelsea.
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