This study considered the optimization of cultural conditions for enhanced lipolytic enzyme activities amongst 4 bacterial Isolates. (Soil samples from different oil-impact soil areas in Ogun -State Nigeria were obtained with soil auger and transported the Biotechnology Laboratory of Federal Institute of Industrial Research Oshodi, Lagos-Nigeria. Soil was subjected to Microbiology studies such as Primary Isolation. Pure cultures obtained test using tributyrin agar, Lipase - hyper producing bacterial Isolates were identified using biochemical tests and Molecular biology tools (Polymerase chain reaction and gene sequencing) out of a total of 11 bacterial Isolates, 4 bacterial Isolates identified as hyper-pro-ducers were Isolate H (Bavibacillus brevis strain HK 544), Isolate A (Pseudommas aeruinosa strain WES 2), Isolate B (Bcillus megaterium strain WH 13), and Isolate F (Bacillus subtilis BS 01) changes in PH of culture/production medium showed that pH 7.0, favoured the Lipase production in Bacillus subtilis (Isolate F) and Pseudomonas aeruginosa (Isolate A) while pH values of 8.0 favoured Lipase production in Isolate H (Brevibacillus brevis). Variation in temperature showed that the 4 bacterial strain responded best to Mesophilic PH ranges from 25 oC- 40 oC. The optimal Nitrogen source as identified in the study was KNO3 and NH4 No3 at higher concentrations (6 g/C). In comparison, Tobacco seed oil (TSO) competed favorably as carbon source with Olive oil as activities of Lipase from the 4 bacterial Isolates ranged from 8.0 - 28.0 units. The use of lesser known oilbased carbon source like Tobacco seed oil has shown a lot of value addition in terms of commercial value.