It was found in this study that the presence of sophorolipid (SL) enhanced the production of short-chain fatty acid (SCFA) from anaerobic fermentation of waste activated sludge (WAS). Experimental results showed that with an increase of SL addition from 0 to 0.1 g/g TSS, the maximal SCFA yield increased from 50.5 ± 4.9 to 246.2 ± 7.5 mg COD/g VSS. The presence of SL reduced the surface tension between hydrophobic organics and fermentation liquid, which thereby accelerated the disintegration of WAS and improved the biodegradability of the released organics. SL promoted the carbon/nitrogen ratio of the fermentation system, enhancing the conversion of proteins in WAS. Moreover, SL suppressed severely the activities of methanogens, probably due to the drop of pH caused by SL addition. Amplicon sequencing analyses revealed that SL increased the abundance of hydrolytic microbes such as Bacteroides sp. and Macellibacteroides sp., and SCFA producers (e.g., Acinetobacter sp.).