Polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene and fluoranthene are commonly found in wastewaters from refineries and biomass gasification industries. This study investigated the simultaneous biodegradation of these PAHs along with lipid accumulation by Rhodococcus opacus in a ternary substrate system. A 23 full factorial design of experiments was employed with the three PAHs at two different levels by varying their initial concentrations in the range 50–200 mg L−1 each. A maximum removal of 91.6%, 82.3% and 80.7% was achieved for naphthalene, phenanthrene and fluoranthene, respectively. The individual effect of PAH concentration was found to be more significant than 2-way and 3-way interaction effects on their degradation. PAH biodegradation efficiency in the mixture was mainly affected by initial concentration and aromatic complexity of the PAHs. Identification of the PAH degradation metabolites was carried out using LC-MS analysis, which clearly revealed that the PAHs were degraded primarily via the ortho/para pathway. This study demonstrates the potential utility of R. opacus for bioremediation and industrial wastewater treatment.