Many organisms produce lactic acid by fermentation, but most industrially important strains are from the genus Lactobacillus and Rhizopus oryzae. L(+)-Lactic acid is the only optical isomer for use in pharmaceutical and food industries because human body is only adapted to assimilate this form. In this research, six strains of Lactobacillus and four strains of R. oryzae (known as high producer) were examined for optical isomers of lactic acid. The production of lactic acid was improved and lactic acid produced in submerged media on rotary shaker incubator. The optical isomers of lactic acid were examined by L(+) and D(-) lactate dehydrogenase kit. All the R. oryzae strains tested produced only L(+) isomer of lactic acid. The highest fungal and bacterial producer strains were R. oryzae PTCC 5263, Lactobacillus plantarum PTCC 1058, L. Bulgaricus PTCC 1332 and L. delbruekii subsp delbruekii PTCC 1333. Lactobacilli strains produced combination of both optical isomers of lactic acid. Among them, L. casei subsp. Casei produced the low amount of D(-)-lactic acid (2%). The optimum concentration of glucose for lactic acid production by R. oryzae and Lactobacillus strains were 180 g/l and 80–120 g/l, respectively.