Oil pollution is one of the most critical environmental pollutions affecting soil's biological, physical, and chemical properties. There are several methods to measure MBC, the most important of which are chloroform fumigation extraction (CFE), chloroform fumigation incubation (CFI), and experimental methods based on substrate-induced respiration (SIR). Due to the presence of high oil content in the oil-polluted soil samples and high content of organic carbon (OC), the application of the CFE method has some limitations because tracking minor changes of MBC in a huge background of OC is not possible. The effect of three levels of oil pollution (L: low, M: moderate, and H: high) on MBC measured by CFI (MBC-CFI) and SIR (MBC-SIR) was investigated through an incubation experiment. Hence, 120 oil-contaminated soil samples were gathered from four selected locations of polluted sites in Naft-Shahr, Kermanshah, Iran. Four experimental formulas including MBC = 16.18×SIR + 5.11 (Eq. 1), MBC = SIR×30 (Eq. 2), MBC = SIR×25.3 (Eq. 3), and MBC = 40.04×SIR + 0.37 (Eq. 4) to estimate the amount of MBC-SIR were used. The results showed that there is a positive and significant correlation between two methods used in this study (r = 0.91, P < 0.01). In both methods, the MBC obtained in H soils was higher than in L and M soils. The values of MBC-CFI data ranged from 264.685 in L soils to 419.459 mgC 100g− 1 in H soils. Among the four used formulas in this study, the lowest amount of RMSE and SI was obtained for Eq. 4. The MBC-SIR measured by Eq. 4 ranged from 290.224 mgC 100g− 1 in L soils to 478.107 mgC 100g− 1 in H soils. According to this study's results, experimental methods based on SIR, especially Eq. 4 can also be used to estimate the amount of MBC in oil-contaminated soils. Moreover, in this study, multiple linear regression analysis and principal components analysis (PCA) was performed on data to get insight into the relation of bio-physical-chemical properties of soil, especially with MBC.