This study was performed to evaluate the potential of acclimated halophilic microorganisms, commercial microorganisms, and microorganisms from polluted soil to degrade crude oil in high salinity oily wastewater (synthetic produced water) at different salt concentrations ranging from zero to 250,000 mg L−1 of total dissolved solids (TDS). The highest degradation of crude oil (>60%) was found for acclimated halophilic microorganisms at TDS of 35,000 mg L−1. An increase in the TDS concentrations above 145,000 mg L−1 leads to a significant decrease in the growth of microorganisms. The results showed that efficiency of the commercial microorganisms was less than the acclimated halophilic microorganisms. The oil biodegradation followed substrate inhibition kinetics and the specific growth rate were fitted to the Haldane model. The biokinetic constants for the saline oily water at TDS of 35,000 mg L−1, i.e., Y, Ks, µmax, and 1/Ki, were 0.21 mg MLSS/mg crude oil, 0.27 mg L−1, 0.019 h−1, and 0.002 mg L−1, respectively.