Biosurfactants are valuable microbial amphiphilic molecules (consisting of molecules having a polar water-soluble group attached to a water-insoluble hydrocarbon chain) with effective surface-active and biological properties applicable to several industries and processes. In recent years, natural biosurfactants had attracted attention because of their low toxicity, biodegradability and ecological acceptability. Two Bacilli species were tested for their abilities to produce biosurfactants by measuring their emulsification activity, emulsification index, oil displacement test, drop collapse and spreading over the blood agar plates. Also, the effects of different carbon and nitrogen sources, as well as, pH and inoculum size were examined as factors affecting the surfactants biosynthesis. Results showed that both Bacilli isolates can produce biosurfactant by using waste frying oil as a carbon source and peptone as a nitrogen source. There was an effect for different factors used in the production of biosurfactants showed by two isolates. Both Bacilli isolated were identified by 16s rRNA as Bacillus subtilis and Bacillus megaterium.