The aim of this study was to investigate the production of succinic acid from lactose concentrate, a by-product of cheese-making, using Actinobacillus succinogenes and Basfia succiniciproducens. Although the ability of these strains to metabolize different sugars is already known, their application in the conversion of lactose bears high potential for optimization. With regard to B. succiniciproducens, this approach is completely novel. In particular, the effect of the medium’s sugar concentration as well as the ability of its supplementation with yeast extract to prevent a lack of essential nutrient proteins and vitamins was examined. Lactose-based media containing sugar concentrations of between 20 and 65 g L⁻¹ and 5 g L⁻¹ of yeast extract were fermented, with both strains showing comparable performances. The best results in terms of succinic acid yield and acid concen-tration—0.57 g g⁻¹ initial sugar and 23 g L⁻¹—were achieved at an initial sugar concentration of 43 g L⁻¹. The necessity of yeast extract was demonstrated using the sugar-optimized medium without supplementation. As a result, the yield and concentration of succinic acid dropped to 0.34 g g⁻¹ and 13 g L⁻¹ and the sugar consumption decreased from more than 99 to less than 55%. Therefore, the supplementation amount of 5 g L⁻¹ of yeast extract can be regarded as well-balanced.