Prospecting for microorganisms and substrates for the synthesis of significant amounts of biosurfactants is becoming more and more prominent due to the rising demand for these substances for use in environmental and industrial applications. This study was carried out to evaluate the effect of different carbon sources on biosurfactant production using bacteria isolated from hydrocarbon-contaminated soil in Hotoro Nasarawa Local Government, Kano State, Nigeria. The physicochemical properties of the hydrocarbon-contaminated soil as well as cultural, morphological, and biochemical characteristics of the isolates were analysed using standard procedures. Methods for screening the isolates for biosurfactant production that include blood haemolysis, drop collapse, oil displacement, and emulsification index tests were employed. The result of physicochemical characteristics of the hydrocarbon-contaminated soil samples from this study revealed that the pH of the all soil samples were weakly acidic from 6.80 to 7.06. The temperature was ranged between 38 and 41 °C. The viable heterotrophic bacterial count of all the samples ranges from 1.56 to 2.78 × 106 cfu/g. Four (4) bacterial genera were biochemically identified as Bacillus subtilis, Bacillus subtilis, Bacillus lichenoformis, and Bacillus sp. The four (4) identified bacterial isolates were all positive for haemolysis test, drop collapse and oil displacement test. The most potent isolate during the screening was Bacillus subtilis which had the highest biosurfactant production ability of 0.83 ± 0.07 and 1–90 ± 0.01 mg/ml for used engine oil and diesel as carbon sources, respectively. It can be concluded that the production of biosurfactant from the diesel substrate was higher than the yield from the used engine oil substrate.