The enzyme activities of the fungus Lasiodiplodia theobromae (L. theobromae) were studied during degradation of benzo[a]pyrene (BaP). The L. theobromae was isolated from a polycyclic aromatic hydrocarbons (PAHs) contaminated soil collected from the Beijing Coking Plant in China and can potentially use BaP as its sole carbon source with a degradation ratio of up to 53% over 10 days. The activities of lignin peroxidase (LiP) and laccase (LAC) could be detected during BaP biodegradation; while manganese peroxidase (MnP) was not detected. Both glucose and salicylic acid enhanced BaP biodegradation slightly. In contrast, the coexistence of phenanthrene (PHE) inhibited BaP degradation. These metabolic substrates all enhanced the secretion of LiP and LAC. The addition of Tween 80 (TW-80) enhanced BaP biodegradation as well as the LiP and LAC activities. At the same time, TW-80 was degraded by the L. theobromae. In addition, the L. theobromae was compared to Phanerochaete chrysosporium (P. chrysosporium), which is a widely studied fungus for degrading PAH. P. chrysosporium was unable to use BaP as its sole carbon source. The activities of LiP and LAC produced by the P. chrysosporium were less than those of the L. theobromae. Additionally, the four intermediates formed in the BaP biodegradation process were monitored using GC-MS analysis. Four metabolite concentrations first increased and then decreased or obtained the platform with prolonged BaP biodegradation time. Therefore, this study shows that the L. theobromae may be explored as a new strain for removing PAHs from the environment.