Background In the industrial biotechnology, ligninolytic enzymes are produced by single fungal strains. Experimental evidence suggests that co-culture of ligninolytic fungi and filamentous microfungi results in an increase laccase activity. In this topic, only the ascomycete Trichoderma spp. has been studied broadly. However, fungal ligninolytic-filamentous microfungi biodiversity interaction in nature is abundant and poorly studied. The enhancement of laccase and manganese peroxidase (MnP) activities of Trametes maxima as a function of time inoculation of Paecilomyces carneus and under several culture conditions using Plackett-Burman experimental design (PBED) were investigated. Results The highest increases of laccase (12,382.5 U/mg protein) and MnP (564.1 U/mg protein) activities were seen in co-cultures I3 and I5, respectively, both at 10 d after inoculation. This level of activity was significantly different from the enzyme activity in non-inoculated T. maxima (4881.0 U/mg protein and 291.8 U/mg protein for laccase and MnP, respectively). PBED results showed that laccase was increased (P < 0.05) by high levels of glucose, (NH4)2SO4 and MnSO4 and low levels of KH2PO4, FeSO4 and inoculum (P < 0.05). In addition, MnP activity was increased (P < 0.05) by high yeast extract, MgSO4, CaCl2 and MnSO4 concentrations. Conclusions Interaction between indigenous fungi: T. maxima-P. carneus improves laccase and MnP activities. The inoculation time of P. carneus on T. maxima plays an important role in the laccase and MnP enhancement. The nutritional requirements for enzyme improvement in a co-culture system are different from those required for a monoculture system.