The removal of antibiotics and resistance genes in wastewater treatment plants has attracted widespread attention, but the potential role of residual antibiotics in the disposal of waste activated sludge (WAS) has not been clearly understood. In this study, the effect of roxithromycin (ROX) on volatile fatty acid (VFA) recovery from WAS anaerobic fermentation was investigated. The experimental results showed that ROX made a positive contribution to the production of VFAs. With the increase of ROX dosages from 0 to 100 mg/kg TSS, the maximum accumulation of VFAs increased from 295 to 610 mg COD/L. Mechanism studies revealed that ROX promoted the solubilization of WAS by facilitating the disruption of extracellular polymeric substances. In addition, ROX enhanced the activity of acetate kinase and inhibited the activities of α-glucosidase and coenzyme F, and showed a stronger inhibitory effect on methane production than the hydrolysis process, thus resulting in an increase in VFA accumulation. These findings provide a new insight for the role of antibiotics in anaerobic fermentation of WAS.