The objective was to evaluate the anaerobic digestion of spent mushroom substrate (SMS) and bovine manure (BM). The experiment was conducted at the Multiuser Research Laboratories, part of the Group of Renewable and Alternative Rural Energies (LabGERAR) at the Federal Rural University of Rio de Janeiro (UFRRJ). Bench-scale Indian model anaerobic digesters were batch-loaded with 1.7 kg of substrate in the proportions based preliminary tests (1:5:0, 1:5:1, and 1:0.1:1 SMS:W (Water):BM). The characterization of the substrates was performed to evaluate the physical–chemical analysis. The substrate temperature profile and the cumulative biogas production potential (L biogas per kg of substrate) were monitored during nine weeks. Analysis of variance followed by Tukey’s test at a 5% probability level was performed. The SMS, as biomass for the anaerobic digestion process, showed a granulometry 20 mesh, extractive content of 6.43%, ash content of 6.7%, lignin content of 22.35%, and holocellulose content of 39.74%. There was no significant difference observed in the volatile solids content, which remained at 11.15%. However, the ash content in bovine manure was three times higher compared to the proportions studied. The substrate temperature remained within the mesophilic range, ensuring lower energy costs and greater process stability. Regarding the cumulative biogas production potential, it was observed that anaerobic digestion between bovine manure and spent mushroom compost, at 1:5:1 SMS:W:BM ratio, showed the highest biogas production potential with faster stabilization. Concludes that SMC possesses ideal characteristics as an anaerobic co-digestant with bovine manure, demonstrating significant potential for fungal biomass conversion.