The reuse of spent A. bisporus substrate for the biodegradation of PAH in multi-polluted soil is feasible. However, the SAS application method is a key factor in obtaining the maximum efficiency. The application of sterilized SAS (SSAS microcosms) does not biodegrade PAH in Pb co-polluted soil despite enhancing the bacterial population. The use of SAS without previous treatment provides certain PAH biodegradation by the inoculation of an appreciable inherent microbiota able to degrade PAH, however this was not feasible as an inoculum carrier for A. bisporus and the heavy pollution
of the soil produced toxic effects on the microbial community. The SAS sterilization and further A. bisporus re-inoculation is adequate as an A. bisporus inoculum carrier and is the most efficient way of SAS reutilization for PAH biodegradation in Pb co-polluted soil by the high activity of A. bisporus. The increment of Pb availability was very low and minimized with time, but the reduction of PAH and its carcinogenic risk is sufficient to consider this method as an effective, cheap and useful soil mycoremediation strategy.