In recent years, the substantial increase in municipal solid waste incineration fly ash (MSWIFA) production has made its treatment a critical issue. However, the high toxicity of MSWIFA makes its utilization still in the exploratory stage. In this study, the heavy metal leaching rate, particle size distribution and activity index of MSWIFA were tested to investigate the detoxification and activation effects of Bacillus mucilaginosus on MSWIFA by introducing rhamnolipid. The results show that the microbial treatment can greatly increase the leaching rate of heavy metals in MSWIFA and its 28-day and 90-day activity indexes, especially by the synergistic treatment. For the Bacillus mucilaginosus and rhamnolipid treated MSWIFA (BR-MSWIFA), the maximum leaching rates follow the order from high to low of 85.57% Cr, 78% Cu, 76.38% Zn, 62.78% Pb and 37.65% Mn, while having a low mass loss of less than 5%, which is important for its reuse. Moreover, compared with the untreated MSWIFA (U-MSWIFA), the average particle sizes of Bacillus mucilaginosus treated MSWIFA (B-MSWIFA) and BR-MSWIFA decrease from 8.39 μm to 7.80 μm and 4.31 μm, and the 28-day activity indexes increase from 80.19% to 101.59% and 110.61%. The effect mechanism is that rhamnolipid not only can promote the growth, reproduction, and metabolic acid production of Bacillus mucilaginosus, but also disperse the extracellular polymeric substance (EPS) and MSWIFA particles, thus increase their contact area and the bioleaching. This study provided a theoretical foundation for the harmless treatment and resource utilization of solid wastes containing heavy metals.