The effects of three widely-used surfactants on waste activated sludge (WAS) fermentation and microbial community structures were investigated. Rhamnolipid bio-surfactants (RL) showed more positive effects on WAS hydrolysis and acidification compared to chemosynthetic surfactants, such as sodium dodecylsulphate (SDS) and sodium dodecyl benzene sulfonate (SDBS). The highest SCOD and VFAs concentrations obtained with RL were 1.15-fold and 1.16-fold that of SDS, and up to 1.73 and 3.63 times higher than those obtained with SDBS. Pyrosequencing analysis showed that an evident reduction in bacterial diversity in surfactant-treated WAS. Moreover, acid-producing bacteria (such as Megasphaera and Oscillibacter), detected with RL, were (6.8% and 6.4% in proportion) more abundant than with SDS, and were rarely found in SDBS and the control. The results also revealed that RL allowed efficient hydrolysis enhancement and was favorable to functional microorganisms for further acidification during WAS fermentation.