AbstractPretreatment with white-rot fungi can effectively remove lignin and decompose the structure of biomass to enhance subsequent enzymatic hydrolysis. This study developed a novel fungal pretreatment of biomass, which was operated under non-sterile conditions. The white-rot fungus Irpex lacteus colonized stably on the non-sterile substrates and effectively degraded lignin. After non-sterile fungal pretreatment for 42 days, lignin was degraded by 43.8%. The maximum sacchariﬁcation efficiency was 7-fold higher after enzymatic hydrolysis compared to that of raw corn stover. Furthermore, the production of ethanol from corn stover improved. During non-sterile biological pretreatment, several microorganisms coexisted with Irpex lacteus, and the microbial community generated abundant by-products that greatly improved the efficiency of enzymatic hydrolysis. Non-sterile fungal pretreatment presents a feasible and promising technology for the production of biofuels by integrating on-farm wet storage systems. Moreover, it provides a low-cost bioconversion process and a stable, secure, and environmentally friendly energy supply.