Biological mannitol production at industrial scale is hindered by the elevated costs of fructose-rich feedstocks. Grape must could be an interesting feedstock for this process, due to its large production and to the commercial imbalance caused by unsold wine stocks in producing countries. The feasibility of employing red must and white must for mannitol production was assessed with Lactobacillus fermentum CECT 285, Leuconostoc mesenteroides CECT 8146 and Lactobacillus intermedius NRRL B-3693. By means of experimental design based on response surface methodology (RSM), it was determined that nutrient supplementation could be limited to the addition of manganese and yeast extract, and that grape must should be diluted with water to improve mannitol production. Under optimal conditions, Lb. intermedius NRRL B-3693 produced 68.9 ± 0.57 g/L mannitol from red must and 79.8 ± 0.25 g/L mannitol from white must in 48 h, attaining yields of 0.888 ± 0.014 and 0.895 ± 0.001 mol/mol, respectively. In order to further reduce costs, yeast extract was replaced by wine lees. Red wine lees (RWL) performed better than white wine lees (WWL) as nitrogen source for mannitol production. Grape musts supplemented with manganese and RWL produced 59.4 ± 0.13 g/L mannitol in the case of red must and 65.6 ± 0.06 g/L mannitol in the case of white must in 144 h with Lb. intermedius NRRL B-3693. Therefore, winery surplus and by-products could be used to produce mannitol, thus opening new biorefinery opportunities for rural areas devoted to winegrowing.