The valorization of agro-industrial byproducts is a key strategy for developing sustainable industries. This study explored the potential of Cucurbita pepo peel, a common waste, as a low-cost fermentation medium for producing Saccharomyces cerevisiae biomass, comparing its performance against the valuable pulp juice. Fermentations were conducted to optimize the initial sugar concentration and evaluate the effect of various nitrogenous sources, including yeast extract, casein hydrolysate, and economical ammonium sulfate mixtures. Results showed an optimal sugar concentration of 10%, where the peel extract produced 2.1-fold more yeast biomass than the pulp juice. While supplementing pulp juice with yeast extract or casein hydrolysate yielded the highest biomass (up to 28.53 g/l), a more economical 1:3 mixture of (NH₄)₂SO₄/yeast extract still achieved a significant 20.24 g/l. Similarly, supplementing the peel extract with yeast extract resulted in a high biomass concentration of 20.83 g/l and a productivity of 0.42 g/l.h . These findings demonstrate that Cucurbita pepo peel is a highly effective and viable substrate, representing a sustainable and cost-efficient alternative to the edible pulp for the industrial-scale production of S. cerevisiae biomass.