An investigation was carried out to compare the ability of two bacteria Pseudomonas aeruginosa PSA5 and Rhodococcus sp. NJ2 isolated from petroleum sludge for degradation of benzo(a)pyrene [B(a)P], a HMW PAH compound in MSM. During 25 days of incubation, 50 ppm B(a)P was degraded by 88 and 47 % by P. aeruginosa PSA5 and Rhodococcus sp. NJ2, respectively. Besides, involvement of different catabolic enzymes, that is, salicylate hydroxylase, 2-carboxybenzaldehyde dehydrogenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, was also examined to identify their differential role in B(a)P degradation. Among these enzymes, the highest induction of 2-carboxybenzaldehyde dehydrogenase (773.5 nmol mg−1 protein) was recorded in P. aeruginosa PSA5, while salicylate hydroxylase was highly expressed (839.6 nmol mg−1 protein) in Rhodococcus sp. NJ2. Both the bacteria were found biosurfactant (glycolipid) producing, and role of biosurfactant in PAH degradation was also ascertained by reduced surface tension, higher emulsification index and increased cell surface hydrophobicity.