The search for alternative sources of energy, such as biodiesel, has been stimulated, since this biofuel is highly susceptible for biodegradation and has low toxicity, thus, reducing the impact in ecosystems. The objective of this study was to select a bacterial consortium with potential for degrading diesel/biodiesel blends (B20) obtained from areas contaminated with hydrocarbons/esters. In order to evaluate the biodegrability of the blend, six enzyme assays were conducted: alkane hydroxylase, Catechol 1,2-dioxygenase, Catechol 2,3-dioxygenase, Protocatechol 3,4-dioxygenase, ρ-NPA hydrolysis (esterase), and release of fatty acids through titration (lipase), with estimative of total protein and biosurfactant production (surface tension measurement and emulsifying index E(24)). The best results obtained allowed the selection of four bacteria isolates (Bacillus megaterium, Bacillus pumilus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia) for compiling a consortium, which will be used for bioaugmentation strategies in soils contaminated with these fuels. This consortium exhibited high potential for biodegradation of biodiesel, and might be an efficient alternative for cleaning up these contaminated environments.