Lactic acid bacteria are widely used because they produce lactic acid naturally, are resistant to acidic pH and a wide temperature range, and frequently produce lactic acid as a primary metabolite. In this study, Enterococcus durans isolated from buffalo milk was employed in lactic acid fermentation with the primary goal of obtaining fermentation parameters for an effective process enabling the use of lactose as an alternative carbon source. Fermentative parameters such as initial concentration of carbon source, dissolved oxygen concentration, cell recycling, and batch with pulse operation mode were studied to find the best conditions for L-(+)-lactic acid production. The association of 20 g·L−1 of lactose with 10 g·L−1 of glucose enabled the best bioconversion to lactic acid. Anaerobiosis did not contribute to increasing lactic acid production. Batch fermentation with cell recycling was the strategy that enhanced lactic acid production and lactose consumption, reaching 26.07 g·L−1, 0.36 g·L−1·h−1 of productivity and yielding about 0.86 g·g−1. It is fundamental to evaluate the parameters of lactic acid fermentation and provide efficient and sustainable production methods.