Abstract. Alyousif NA, Al-Tamimi WH, Al-sahib MAA. 2022. Evaluation of the effect of various nutritional and environmental factors on biosurfactant production by Staphylococcus epidermidis. Biodiversitas 23: 3533-3538. Biosurfactants are biological surface-active compounds synthesized mainly by hydrocarbon-utilizing bacteria. The properties of biosurfactants make them promising compounds for the application in various fields. The current study evaluated the effect of various nutritional and environmental factors on biosurfactants produced by Staphylococcus epidermidis. This bacterium, for first time in world was isolated from oilfield reservoir in the current study and identified by 16S rDNA sequencing and considered biosurfactant producer according to screening tests. Several factors were evaluated in the current study to assess the optimal conditions for producing the biosurfactant. The results demonstrated that the best carbon source was olive oil with 2% concentration and glutamic acid was the best nitrogen source with 0.2% concentration. The study demonstrated that the best biosurfactant production was recorded at incubation time of 5 days with a 3% inoculum size. Further study is required to determine the chemical structure of produced biosurfactants and to evaluate the potential application of biosurfactants in various fields, therefore constituting a stimulus for future studies.