Acid oil produced from the acidulation of soapstock was investigated as a feedstock for producing a glycolipid biosurfactant, manosylerithritol lipid (MEL), by microbial fermentation. Fermentation with the optimized acid oil substrate concentration of 200 g L−1 resulted in 3.89 g L−1 of MEL. To enhance productivity, the titer MEL was produced by a multistage fermentation process with the periodic addition of 20–50 g L−1 substrate after the stationary phase of the culture. The repeated substrate feeding marginally enhanced the MEL titer up to 5.56 g L−1. The MEL from the culture broth was recovered by solvent extraction at different pH levels. Among the solvents tested, ethyl acetate: acetonitrile (9:1) demonstrated a higher partition coefficient value of 3.1 in acidic conditions with 75.6% MEL recovery. The MEL produced by using acid oil as feedstock has significant surface-active properties, measuring 2.8 × 10−6 M in critical micelle concentration with 29.3 mN m−1 surface tension.