This work aimed to evaluate the potential of three fungi strains, Lentinula edodes, Pleurotus eryngii, and Pleurotus citrinopileatus, to degrade lignin and enhance the nutritive value of grape stalks (GS). The GS was inoculated with the fungi and incubated under solid-state fermentation at 28 °C and 85% relative humidity for 7, 14, 21, 28, 35, and 42 days, in an incubation chamber. The influence of the treatments was evaluated by analyzing the potential modifications in the chemical composition, in vitro organic matter digestibility (IVOMD) and enzymatic kinetics. An increase (p < 0.001) in the crude protein content was observed in the GS treated with L. edodes and P. citrinopileatus at 42 days of incubation (50 and 75%, respectively). The treatment performed with L. edodes decreased (p < 0.001) lignin content by 52%, and led to higher (p < 0.001) IVOMD values at 42 days of incubation. By contrast, P. eryngii did not affect lignin content and IVOMD. A higher activity of all enzymes was also detected for the treatment with L. edodes. Results indicated that L. edodes has a great potential to enhance the nutritive value of GS as an animal feed, due to its lignin degradation selectivity.