Aims A culture medium based on apple bagasse was designed and tested as a substrate for biomass production of conventional and unconventional native wine yeasts. Methods and Results The physicochemical characterization of the apple bagasse was carried out and its potential utility as a constituent of a complete culture medium for the production of yeast biomass was analysed using the experimental statistical designs. Growth parameters of conventional and nonconventional Patagonian wine yeasts were analysed with Placket-Burman designs and response surface methodology, comparing in each assay the apple bagasse substrate with the commonly used substrate for biomass development, cane molasses. Culture media composition was optimized and models were validated. Conclusions This study demonstrates that, both from a nutritional and from an economic point of view, apple bagasse constitutes a more advantageous substrate than cane molasses for the propagation of native yeasts from Patagonia. Significance and Impact of the Study We used an alternate carbon-rich material, generously available in our region, originally generated as fruit industrial waste, to transform it into a source of sustainable, economically profitable and environmentally friendly energy resource.