Argentina midfielder Mascherano, who only came on as a late substitute for Steven Gerrard in Saturday's 6-1 hammering of Hull City, is suffering from sickness, while Israel international Benayoun picked up a leg injury in the same match. Neither travelled with the party on Monday.
Boss Rafael Benitez may well have opted for a more cautious approach against Fiorentina had Mascherano been available, but it is now likely that that winger Albert Riera will stay in the side.
That will be good news for deadly strike duo Gerrard and Fernando Torres who arrived in Florence in goalscoring form and hailed each other as "the best in the world" ahead of the match.
The duo have struck up a great partnership in attack for the Reds and in the 77 games the pair have played together they have plundered 86 goals between them - Gerrard with 39 and Torres 47.
Gerrard said: "I wouldn't swap Fernando for any other striker in the world, he's the best."
And Torres added: "If you play alongside someone like Stevie, who is the best in the world, it can only help you. He is a great passer and can score goals."
Fiorentina will be well aware that the pair have a pretty impressive Champions League record. In the 15 Champions League games they have played together, they have scored 15 goals.
Meanwhile, Liverpool have confirmed that defender Daniel Agger will make his return from injury in Tuesday night's reserve tie against Manchester City. Agger has not featured this season due to a back injury but his imminent return is welcome news indeed for Benitez.
"It is fantastic to see Daniel back and it's a great boost for everyone at the club," reserve manager John McMahon told the club's official website. "It's always great to see quality players come back from injury and this will be a perfect game for him to make his comeback.
"It's a fantastic stadium with a great pitch and it is always a fantastic boost for the young lads when they have a top professional like Daniel playing alongside them. The young lads look up to him and they can learn from him as well."