Planted forests consist of areas of wood cultivation destined for various commercial applications. The remaining stumps and roots constitute obstacles to the passage of machines and implements to news cycles of cultural management. To overcome the disadvantages of mechanical stump removal, this study aimed to prospect and analyze the lignocellulolytic enzyme profile of 11 fungi isolated from wood and evaluate their ability for in vitro eucalyptus chips degradation. The fungi were cultivated in semi-solid media in 5 different biomasses, which were chemically characterized, for 5 and 8 days. The fungi Hypoxylon sp. and Aspergillus sydowii demonstrated greater activities for some enzymes, while Chrysoporthe cubensis demonstrated interesting activities for most of them, such as 1.3, 2.8, and 1.6 U.mL−1 for FPase, endoglucanase, and β-glucosidase, respectively. The enzymatic extract of C. cubensis, selected for Eucalyptus sp. wood chips hydrolysis, at 20 FPU.g−1 of chip, promoted an increase of reducing sugars to up to 160.3 mmol.L−1 in 10 days. Moreover, it released higher glucose (17.6 g.L−1) and xylose (7.6 g.L−1) amounts compared to the commercial cocktail. Microscopic analysis suggested changes in the structure and constituents by enzymes, which confirms the potential of C. cubensis enzymatic extract for wood degradation.