Glycerol from spent oil was processed by transesterification for biodiesel production. Although glycerol contains many types of impurities, it can be used as a C-source for lactic acid production by fungi, such as Rhizopus microsporus. In this study, we found that wild type R. microsporus (LTH23) produced more lactic acid than the mutant strains on cabbage glycerol media (CG media). More lactic acid was produced on CG media than on cabbage extract media (C media) by about two-fold in batch fermentation conditions. In addition, we found that lactic acid production in a fed-batch process was also slightly higher than in a batch process. To study the combined effects of pH, urea, and glycerol waste concentration on lactic acid production, a response surface methodology was used. The optimum pH, urea, and glycerol waste concentrations were pH 6.5, 3.75g/L, and 17g/L, respectively. The maximum lactic acid production predicted by this equation model was 4.03g/L.