Abstract Efficient degradation of lignocellulosic biomass is key for the production of value-added products, contributing to sustainable and renewable solutions. This study employs a two-step approach to evaluate lignocellulolytic enzymes of Ceratocystis paradoxa, Colletotrichum falcatum, and Sporisorium scitamineum. First, an in silico genomic analysis was conducted to predict the potential enzyme groups produced by these fungi. Second, a 2³ full factorial design of solid-state cultivation was employed to investigate the cultivation conditions that optimize enzyme activity. In silico analysis of phytopathogen genomes identified proteins with the potential for biomass degradation. Cellulase and phenoloxidase activities were assessed in culture medium and solid-state cultivation. A 2³ full factorial design was employed for solid-state cultivation to evaluate the cellulose, endoglucanase, and laccase activities. In silico analysis shows that C. falcatum has the most diverse enzyme set for lignocellulosic biomass degradation. In vitro assays corroborate this, demonstrating that C. falcatum produces the highest enzyme quantities, except for cellulase, where C. paradoxa outperforms it. Both C. paradoxa and C. falcatum exhibit cellulase and phenoloxidase activities, but only C. falcatum shows laccase activity. Most favorable enzyme production in solid-state cultivation occurred with 85–95 g 100 g− 1 bagasse moisture and 5 g 100 g− 1 yeast extract, with four-day cultivation period needed for cellulase and endoglucanase in C. paradoxa and 12 days for endoglucanase and laccase in C. falcatum. The in silico and in vitro assays demonstrated that C. falcatum can produce a diverse enzyme set, including laccase, cellulase, and endoglucanase, making it a promising candidate for enzymatic industrial applications.