Objective To biodegrade the diesel pollution in aqueous solution inoculated with Mycobacterium and filamentous fungi.Methods Bacteria sampled from petroleum hydrocarbons contaminated sites in Karamay Oilfield were isolated and identified as Mycobacterium hyalinum (MH) and cladosporium. Spectrophotometry and gas chromatography (GC) were used to analyze of the residual concentrations of diesel oil and its biodegradation products. Results From the GC data, the values of apparent biodegradation ratio of the bacterial strain MH to diesel oil were close to those obtained in the control experiments. Moreover, the number of MH did not increase with degradation time. However, by using n-octadecane instead of diesel oil, the real biotic degradation ratio increased to 20.9% over 5 days of degradation. Cladosporium strongly biodegraded diesel oil with a real degradation ratio of up to 34% after 5 days treatment. When the two strains were used simultaneously, a significant synergistic effect between them resulted in almost cornplete degradation of diesel off, achieving a total diesel removal of 99% over 5 days of treatment, in which one part of about 80% and another part of about 19% were attributed to biotic and abiotic processes, respectively. Conclusion The observed synergistic effect was closely related to the aromatics-degrading ability of Cladosporium, which favored the growth of MH and promoted the bioavailability of diesel oil.