The Argentine World Cup-winning captain was again absent through injury and was sorely missed in a 4-1 defeat to Chicago Fire.
Former Liverpool, Bayern Munich and Stoke attacker Xherdan Shaqiri helped inflict the painful loss, scoring twice, with his fellow Swiss Maren Haile-Selassie did the same.
Miami are now second-bottom of the eastern conference, and five points from the final available qualifying spot with three games left to play.
Even with a game in hand, the David Beckham-owned side now have little control over their destiny, and could well miss the play-offs for the second time in their four-year history.
Failing to make the top nine would come as a huge blow, particularly considering the team's form since the arrival of Messi in June.
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The Barcelona legend left Paris Saint-Germain on a free transfer and instantly lit up American soccer with goals and assists, as well as a Leagues Cup triumph, the team's first-ever trophy win.
However, alongside his MLS exploits, Messi has continued to lace-up for Argentina, and scored an all important match-winner against Ecuador in World Cup qualifying on September 8.
Since then, though, injury has kept him largely on the sidelines, featuring in just one of a possible seven games for Miami, a 4-0 win over Toronto.
But without their superstar no.10, the Florida side are on a four-game winless run, and heading for a sorry end to the season.