Introduction Naphthalene catabolic strain Pseudomonas putida ND6 harbors two megaplasmids pND6-1 and pND6-2; naphthalene-degrading associated genes are located on pND6-1, while plasmid pND6-2 possesses potential coding genes for conjugative transfer. Methods In this study, the recombinant ND6 in which pND6-1 and pND6-2 were labeled with gentamicin and kanamycin-encoding genes, respectively, was constructed to investigate the conjugative transfer behavior of the two plasmids in distinct aqueous matrices. Results The results indicated that both pND6-1 and pND6-2 plasmids could transfer from donor strain ND6 to a recipient strain P. putida KT2440, while the transfer frequency of pND6-2 (1.90 x 10(-2)) was significantly higher than that of pND6-1 (3.12 x 10(-9)). Furthermore, the concomitant transfer of pND6-1 and pND6-2 was confirmed at a lower frequency of 10(-9) colonies per recipient similar with that of pND6-1. The conjugative transfer efficiency was obviously affected by the initial inoculum and the stability of microbes. Moreover, more than 90% of the transconjugants lost the plasmids after 20 generations cultivation without resistance pressure, suggesting the importance of selective pressure to maintain the plasmid stability. Finally, the naphthalene degradation analysis by mixed ND6 and KT2440 revealed that conjugative transfer of catabolic plasmids contributed to the rapid dispersion of the degradation genes on plasmids and enhanced the naphthalene removal.