Repeated and high-dose carbendazim applications have caused serious soil carbendazim contamination, and eco-friendly and economical approaches have been suggested to promote carbendazim removal in agricultural soil. Spent mushroom substrate (SMS) is a special recycled resource after harvesting mushrooms and can be utilized in contaminated soil amendment. The SMS application into agricultural soil might increase antibiotic resistance gene abundances, and the health risks of SMS application might be reduced with reasonable management to adjust the related electron transport of soil nitrification or denitrification. In this study, the SMS and nitrification inhibitor dicyandiamide were used to remediate agricultural soil contaminated with the carbendazim, and the carbendazim contents, soil microbial biomass, activities and community and human disease genes were determined. Compared to the control treatment, the combined applications of SMS and dicyandiamide significantly decreased soil carbendazim content by 38.14% but significantly enhanced soil β-glucosidase, chitinase, arylsulfatase, urease and electron transfer system activities. The relative abundances of Proteobacteria and Actinobacteria were increased by 11.0% and 8.2% with the SMS application, respectively. The carbendazim residues were negatively correlated with the soil pH, electron transfer system activities and relative abundances of Proteobacteria and Actinobacteria. The relative abundances of human disease genes were also dramatically increased with the SMS application, but compared to the SMS alone, extra dicyandiamide application significantly reduced the relative abundances of human disease genes in soils. The SMS applications into fungicide-contaminated soils could generate double-edged sword effects of facilitating fungicide dissipation but leading to potential health risk increase, while applying the dicyandiamide with SMS might be an effective strategy to decrease the negative effect of health risk.