This study was aimed to assess the plausible anti-obesity effects of Bacillus subtilis SPB1 crude lipopeptide biosurfactant on high fat high fructose diet-fed rats (HFFD). Male Wistar rats were divided into five groups with the following treatment schedule: normal diet (CD), HFFD, HFFD supplemented with SPB1 biosurfactant from the first day of the experiment (HFFD+Bios1, 10mg/kg/day), HFFD receiving standard drug (HFFD+Torva, 10mg/kg/day) or SPB1 biosurfactant (HFFD+Bios2, 10mg/kg/day) during the last 4 weeks of the study. The results showed an increase in body weight of HFFD by ∼19% as compared to controls (CD). Moreover, serum lipase activity underwent a threefold increase which led to an increase in the levels of total cholesterol (T-Ch), triglycerides (TG) and LDL-cholesterol (LDL-Ch) in serum of untreated HFFD, as well as a rise in the calculated atherogenic index (AI). Furthermore, liver dysfunction indices such as AST, ALT, CPK, LDH, GGT, ALP and T-Bilirubins exhibited remarkable increases in serum of HFFD as compared to controls (CD). Whereas, the administration of Bacillus subtilis SPB1 biosurfactant to HFFD improved the body weight gain and serum lipids profile and reverted back near normal the activities of lipase and liver toxicity indicators. In addition, notable protective and curative effects were reported in liver tissues. Overall, these results suggest that the lipopeptides biosynthesized by Bacillus subtilis SPB1 achieved an anti-obesity effect through the inhibition of lipid digestive and liver dysfunction enzymes.