[Objective] The role of natural mediators in oxidation of anthracene and pyrene by laccase producing fungi were evaluated. [Methods] In this work, enzyme activities assay and nondenaturing PAGE were employed to analyze laccase activity. [Results] The results showed that the fungus Pycnoporus sanguineus Z-1 and Fomes fomentarius Z-5 produced laccase, with maximal production of 11.90 and 4.83 U/mL, respectively, but not lignin peroxidase and manganese peroxidase. However, the culture fluid of Fomes fomentarius Z-5, with lower laccase activity, oxidized 74.3% of anthracene and 12.4% of pyrene, higher than that of Pycnoporus sanguineus Z-1, which suggested that the natural mediators might exist in the fungal culture and influenced the anthracene and pyrene oxidation. A further experiment demonstrated that all the treatments with addition of ultrafiltrate, boiled ultrafiltrate or boiled culture fluid improved the antracene and pyrene oxidation. The enhancement levels of ultrafiltrate, boiled ultrafiltrate and boiled culture fluid from Fomes fomentarius Z-5 were higher than those of Pycnoporus sanguineus Z-1, implying that the natural mediators in Fomes fomentarius Z-5 culture was more efficient in improving PAHs oxidation than in Pycnoporus sanguineus Z-1 culture. [Conclusion] The findings indicated that natural mediators played a important role in oxidation of substrates by laccase producing fungi and these might account for the phenomenon that Fomes fomentarius Z-5 culture, with lower laccase activity, oxidized more anthracene and pyrene than Pycnoporus sanguineus Z-1 culture.