This study presents the improved biodegradation of crude oil in aqueous phase using mutant Dietzia sp. obtained by random mutagenesis of wild Dietzia sp. using 60Co-γ irradiation. The mutants obtained were screened based on their degradation performance and the best mutant was selected for oil degradation optimization research. A four factor central composite design coupled with response surface methodology was applied to evaluate and optimize the important variables. A genetically stable mutant, designated as M22, was isolated and demonstrated significantly higher degradation efficiency (52.5%) of total petroleum hydrocarbons (TPHs) than the parental strain (28.2%) in liquid media after 14 days of incubation. Increased production of enzyme responsible for the degradation was achieved with the mutant species. Optimum conditions were determined to be pH 7.6, 0.20 g/L K2HPO4, 0.57 g/L NH4NO3, and 0.62 g/L yeast extract. Approximately 68.5% of TPH was experimentally degraded after 14 h of incubation under the optimum conditions, which agreed well with the model prediction. Gas chromatography-mass spectrum analysis showed that the mutant M22 could degrade a wide range of crude oil fractions, while optimization of culture conditions could be effective for increasing its strain's degrading ability.