Mycobacterium aromativorans JS19b1(T) was isolated from a petroleum-contaminated site that was acclimated almost 100 years. In the present study, metabolism of several polycyclic aromatic hydrocarbons (PAHs) and structural analogues in JS19b1(T) was studied. The proteomic profiles were compared when JS19b1(T) was cultured in nutrient broth and glucose-, phenanthrene-, and phthalate-supplemented mineral media. Proteomic analysis showed notable characteristics of this species, for instance, the existence of enzymes for degradation of multiple classes of chemicals including biphenyl, phenanthrene, dibenzothiophene, and organophosphorus pesticides. Phenanthrene degradation enzymes were detected only in phenanthrene-fed cells, suggesting a very tight regulation of the enzymes. Detection of the other enzymes under various treatment conditions indicated that their regulation may be through very complex mechanisms. In comparison with common major metabolites, PAH transformations produced various types of potentially toxic intermediates, including epoxide, quinone, phenols, aldehydes, and phthalates. In a bioenergy production aspect, PAH transformation does not seem to provide substrates for glycolysis and pentose phosphate pathways. This study signifies the potential of protein profiling for studies of relatively uncharacterized bacteria for biodegradation of environmental pollutants.