Development of bioprocess routes for production of value-added materials from food waste is very attractive in terms of bioprocess economics and environmental protection. In this study, production of 2-keto-d-gluconic acid, a significant platform chemical, from food waste was performed by Pseudomonas reptilivora NRRL B-6 for the first time. The nitrogen source preference of the strain for 2-keto-d-gluconic acid production under different glucose concentrations in synthetic medium has been elucidated. At higher glucose concentrations, utilization of organic nitrogen source (yeast extract) led to higher product formation while at lower glucose concentration, utilization of inorganic nitrogen source (ammonium chloride) was also very feasible. The results indicated that favorable nitrogen source for 2-keto-d-gluconic acid production can change depending on the glucose concentration. The waste bread hydrolysate (glucose 181.43 g/L, protein 1.21% (w/v)) prepared by sequential application of alpha -amylase, amyloglucosidase, and protease was utilized in medium formulation at different glucose concentrations. Bread hydrolysate without additional supplements provided higher product formation than the optimum medium prepared with the hydrolysate. Utilization of 50 g/L CaCO3 led the maximum product formation. The maximum 2-keto-d-gluconic acid production from waste bread was 142.81 g/L with the productivity of 3.02 g/L/h and molar yield of 0.95. The proposed approach in this study confirms that waste bread as a sole source of nutrients can be valorized for microbial production of various industrially relevant platform chemicals.