The study aims to reveal the potency of biosurfactant-producing bacterial culture with molasses as substrate growth in releasing oil from the petroleum sludge at temperature variations. Bacteria used consisted of (Acinetobacter sp. P2(1), Pseudomonas putida T1(8), Bacillus subtilis 3KP and Micrococcus sp. L II 61). The treatments were tested at 40°C, 50°C and 60 °C for 7 days of incubation. Synthetic surfactant (Tween 20) was used as a positive control and molasses as a negative control. Release of petroleum hydrocarbons from oil sludge was expressed in percentage of oil removal from oil sludge (%). Data were analyzed statistically using the Analysis of Variance (α = 0.05) and continued with Games-Howell test. The kinds of bacterial cultures, incubation temperature and combination of both affected the percentage of oil removal. The abilities of Bacillus subtilis 3KP and Micrococcus sp. LII 61cultures in oil removal from oil sludge at the temperature exposure of 60°C were higher than Tween 20. Both of bacterial cultures grown on molasses can be proposed as a replacement for synthetic surfactant to clean up the accumulation of oil sludge in a bottom of oil refinery tank.