The aim of the study was to investigate petroleum waste remediation and toxicity reduction by five bacterial strains: Ralstonia picketti SRS (BP-20), Alcaligenes piechaudii SRS (CZOR L-1B), Bacillus subtilis (I'-1a), Bacillus sp. (T-1), and Bacillus sp. (T'-1), previously isolated from petroleum-contaminated soils. Petroleum hydrocarbons were significantly degraded (91%) by the mixed bacterial cultures in 30 days (reaching up to 29% in the first 72 h). Similarly, the toxicity of the biodegraded petroleum waste decreased 3-fold after 30 days. This work shows the influence of bacteria on hydrocarbon degradation and associated toxicity, and its dependence on the specific microorganisms present. The ability of these mixed cultures to degrade hydrocarbons and reduce toxicity makes them candidates for environmental restoration applications at other hydrocarbon-contaminated environments.