The CDU has been ousted from the state governing coalition with the center-left Social Democrats.
Meanwhile the right-wing anti-migrant party Alternative for Germany (AfD) made gains and will enter the state parliament for the first time.
Angela Merkel’s popularity has waned since her 2015 decision to allow more than a million refugees into Germany.
The CDU won 17.6% of the vote – its worst-ever result in Berlin.
It is the party’s second electoral blow in two weeks, having been pushed into third place by the AfD in Mecklenburg-Western Pomerania at the beginning of the month.
The Social Democrat Party (SPD) has emerged as the strongest party with about 22%, in spite of losing almost 7% of their voters.
The strong showing of the AfD, 14%, has prompted its co-chairman Joerg Meuthen to say the party was strongly positioned for 2017 national elections.
The AfD is now set to be represented in 10 out of 16 state parliaments.
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