This study explores the use of thermophilic biocatalysts, specifically the strain W. coagulans MA-13, for the one-step conversion of citrus waste into lactic acid (LA). The research aims to utilize untreated citrus waste as a sustainable feedstock for LA production through microbial fermentation. The study combines the thermophilic enzymatic cocktail Cellic CTec2 with the hydrolytic capabilities of MA-13 to enhance biomass degradation by up to 62%. Batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into LA, achieving a concentration of up to 44.8 g/L. These results indicate that MA-13 is a promising microbial cell factory for one-step LA production, combining cost-effective saccharification with MA-13's fermentative performance on agri-food wastes. The study also highlights the potential of this approach for the sustainable valorization of agricultural waste streams. The research shows that MA-13 can efficiently produce LA from citrus waste without the need for chemical pretreatment, making it an eco-friendly and sustainable option. The study also identifies the enzymatic activities of MA-13, including the presence of a GH capable of hydrolyzing α-1,6 linkages, and demonstrates the effectiveness of MA-13 in degrading various polysaccharides. The results indicate that MA-13 is a suitable microorganism for the valorization of agri-food wastes for the production of valuable compounds. The study also highlights the potential of citrus waste as a rich source of bioactive compounds with antioxidant, anti-inflammatory, and anticancer properties. The findings suggest that MA-13 is an excellent model system for waste valorization, providing opportunities for sustainable utilization and the generation of value-added products.This study explores the use of thermophilic biocatalysts, specifically the strain W. coagulans MA-13, for the one-step conversion of citrus waste into lactic acid (LA). The research aims to utilize untreated citrus waste as a sustainable feedstock for LA production through microbial fermentation. The study combines the thermophilic enzymatic cocktail Cellic CTec2 with the hydrolytic capabilities of MA-13 to enhance biomass degradation by up to 62%. Batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into LA, achieving a concentration of up to 44.8 g/L. These results indicate that MA-13 is a promising microbial cell factory for one-step LA production, combining cost-effective saccharification with MA-13's fermentative performance on agri-food wastes. The study also highlights the potential of this approach for the sustainable valorization of agricultural waste streams. The research shows that MA-13 can efficiently produce LA from citrus waste without the need for chemical pretreatment, making it an eco-friendly and sustainable option. The study also identifies the enzymatic activities of MA-13, including the presence of a GH capable of hydrolyzing α-1,6 linkages, and demonstrates the effectiveness of MA-13 in degrading various polysaccharides. The results indicate that MA-13 is a suitable microorganism for the valorization of agri-food wastes for the production of valuable compounds. The study also highlights the potential of citrus waste as a rich source of bioactive compounds with antioxidant, anti-inflammatory, and anticancer properties. The findings suggest that MA-13 is an excellent model system for waste valorization, providing opportunities for sustainable utilization and the generation of value-added products.