The present study showed that syntrophic associations in a defined bacterial consortium, named OPK, containing Mycolicibacterium strains PO1 and PO2, Novosphingobium pentaromativorans PY1 and Bacillus subtilis FW1, led to effective pyrene degradation over a wide range of pH values, temperatures and salinities, as well as in the presence of a second polycyclic aromatic hydrocarbon (PAH). Anthracene, phenanthrene or fluorene facilitated complete pyrene degradation within 9 days, while fluoranthene delayed pyrene degradation. Interestingly, fluoranthene degradation was enhanced in the presence of pyrene. Transcriptome analysis confirmed that Mycolicibacterium strains were the key PAH-degraders during the cometabolism of pyrene and fluoranthene. Notably, the transcription of genes encoding pyrene-degrading enzymes were shown to be important for enhanced fluoranthene degradation. NidAB was the major initial oxygenase involved in the degradation of pyrene and fluoranthene mixture. Other functional genes encoding ribosomal proteins, an iron transporter, ABC transporters and stress response proteins were induced in strains PO1 and PO2. Furthermore, an intermediate pyrene-degrading Novosphingobium strain contributed to protocatechuate degradation. The results demonstrated that synergistic interactions among the bacterial members (PO1, PO2 and PY1) of the consortium OPK promoted the simultaneous degradation of two high molecular weight (HMW) PAHs.