Background and aimsSpent mushroom substrates (SMSs) are widely used as organic fertilizer due to their high nutrients. However, the contribution of different types and amounts of SMSs to soil nutrients and microbial communities has been underreported. This study aims to provide data support for the selection and suitable application of SMSs, and to inform sustainable agricultural development.MethodsThe present study conducted a two-year field observation to investigate the effects of substituting 25% or 50% of chemical fertilizer (based on Nitrogen application) with Flammulina velutipes residue (FVR), Agaricus bisporus residue (ABR), and Auricularia auricula residue (AAR) on soil nutrients and microbial communities.ResultsCompared to chemical fertilizer treatment (CF), SMS treatments significantly increased soil organic carbon (OC) and carbon-nitrogen ratio (C/N) by 13.75-27.15% and 16.17-25.25%, respectively. The AAR treatment exhibited superior performance to other SMS treatments in improving soil nutrients. Notably, using AAR to replace 25% of chemical fertilizer (AAR1) was more effective than replacing 50% (AAR2) in enhancing nutrient availability. Compared to CF, the application of SMS significantly enhanced bacterial alpha-diversity without obviously affecting fungi. The relative abundance of Actinobacteria in AAR1 significantly decreased compared to CF. Furthermore, the application of SMS atered the proportions of bacterial and fungal functional groups. Finally, the application of SMS improved microbial diversity and altered microbial community composition by enhancing soil OC and C/N (especially AAR1).ConclusionsThis study crucially provides information on the application of SMS for nutrient amelioration and microbial community modulation, fostering a sustainable and resilient soil ecosystem.