The former captain, out since mid-February, was closing in on a first-team return when his calf muscle gave way as he trained on Sunday.
And manager Arsene Wenger admitted that the club's impatience to bring back their influential central defender could have backfired.
Injury blow: Key defender William Gallas could miss the next month and will not play against Porto
Gallas was sent for a scan on Monday, as was Cesc Fabregas, who hasalso been ruled out of Tuesday's Champions League last-16 tie againstPorto with another hamstring strain, his third in the same muscle sinceearly December.
Wenger is confident Fabregas will not be a long-term absentee, back at the latest to face West Ham a week on Saturday, but his assessment of Gallas was much gloomier.
He said: 'He was feeling very well. He had no pain at all, then he goes for a little jog and boom, it goes again. Unfortunately, we are at the starting point again.
'At the beginning it was a minor injury. We were impatient and we pushed him - he wanted to get on as well - and that is maybe why it has happened.'
The injury to Gallas means Sol Campbell is expected to line up at the back alongside Thomas Vermaelen, but at 35 he is not able to play every game.
With Johan Djourou ruled out for the season with a knee injury, and Philippe Senderos on loan at Everton, Wenger's only other central defender is 32-year-old Mikael Silvestre, who was exposed against Burnley on Saturday, but will have to play some games to ease the workload on Campbell.
Gallas and his representatives are due to meet Arsenal officials later this week for a further round of negotiations about his contract, which expires in the summer.
Wenger wants to keep the 32-year-old defender, who was in outstanding form before injury hit and would like the club to bend their rule of awarding only one-year contract extensions to players over 30.
With Roma manager Claudio Ranieri interested in signing a player he worked with at Chelsea, Gallas is in a strong negotiating position despite his fitness issues.
Campbell's return is ironic as Arsene Wenger's starlets seek inspiration from another old-fashioned central defender who once braved local hatred to make the switch from Tottenham to Arsenal.
The turncoat in question is Willie Young, a name unlikely to even register with most of Wenger's youthful squad.
Not since Young scored Arsenal's winner against Hajduk Split at Highbury to win a UEFA Cup second-round tie on away goals more than 30 years ago have they overturned a first-leg deficit in Europe.
They have never done it in the Champions League, but must start now if they are to progress to the last eight.
'We have a good chance to make history,' smiled Arsene Wenger, who's team trail Porto 2-1 after a first leg most memorable for the mistakes of stand-in goalkeeper Lukasz Fabianski.
'To score away from home is still a massive advantage in the UEFA rules and that is why we are confident. To lose 1-0 away is a terrible result because you know, if you concede one, it's nearly over.
Sol survivor: Campbell will start alongside Thomas Vermaelen for the crunch visit of the Portuguese side
'We have scored and that to me is a massive difference. This time, if you score one, you are through if you don't concede. It is a tricky game but we know we can score goals. We want a better defensive performance.'
Arsenal leaked just one goal in their group games but their defensive frailties were laid bare by Jesualdo Ferreira's Porto in the Estadio do Dragao three weeks ago.
Enlarge Fabianski spilled a low cross into his own net after 11 minutes, before a bizarre mix-up between the Polish keeper and Campbell presented Falcao with a simple winner from a quickly taken free-kick.
'I don't want to talk about a teammate's mistakes,' said Manuel Almunia.
'We had a bad result but everyone is 100 per cent convinced we can do it. Our priority will be to maintain a clean sheet.'
That defensive focus will dominate Wenger's thoughts but he added: 'We want to win this game but we also need to be patient. Maybe it is more important to keep your head.'
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Fabregas's scan showed no major muscle damage but Wenger will take no chances.
He said: 'To play again would mean significant damage. We cannot take a gamble.'
Porto have problems of their own. They required two late goals to salvage a 2-2 draw at Olhanense on Saturday, and Fernando, their Brazilian midfield anchor, is injured and there is a theory experienced boss Ferreira may omit his prolific centre forward Falcao in order to field a more industrious front three.
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