The objective of this study was to evaluate the production (P) (g of fresh mushrooms /bag) and biological efficiency (BE) (g of fresh mushrooms per 100 g of dry substrate) of 17 fungal strains, namelyPleurotus ostreatusPo-IAP, Po-P38, Po-P14, Po-IE202, Po-Sfco, Po-JP, Po-Psma, and Po-POS,Pleurotus djamourPd-PRO and Pd-UTMR,Pleurotus eryngiiPe-MB and Pe-PQ,Lentinula edodesL15, L9, L5, and LC, andHericium erinaceusHeri, produced in corn stover (CS) and to assess the content of crude protein (CP), lignin (L), cellulose, hemicellulose, acid detergent fiber (ADF), and neutral detergent fiber (NDF) andin vitrodigestibility of dry matter (IVDMD) of the residual substrate of CS, the so called spent mushroom substrate (SMS), in comparison to the non-inoculated substrate (C). The variables were analyzed as a completely randomized block design using R 4.0.3 software. Means were compared using Tukey’s procedure. ThePleurotusspp. strains, compared toLentinulaspp. andHericiumspp., presented better BE and P. In comparison to C, the SMS increased the CP content (p < 0.05) from 10.8% (Po-JP) to 70.3% (LC), while NDF decreased (p < 0.05) from 11.5% (Pd-Pro) to 33.5% (L15) and IVDMD increased (p < 0.05) from 16.2% (Heri) to 47.7% (Pd-UTMR). In conclusion, of the 17 strains evaluated, the 3 strains ofLentinula edodes(L5, L15, and L9), one strain ofPleurotus djamour(Pd-UTMR), and one strain ofPleurotus ostreatus(Po-IAP) generated a SMS that, due to its nutritional improvement and increase in IVDMD, could be used as feed for ruminants. Our results also showed that corn stover is a suitable substrate to producePleurotusspp. fruiting bodies, with strain Po-IAP as the best yielding.