Cellulose nanostructures, particularly bacterial nanocellulose (BNC), have gained significant attention due to their superior mechanical strength, biodegradability, and biocompatibility, making them promising alternatives to petrochemical-based plastics. However, large-scale BNC production is limited by low yields and high processing costs, with culture media contributing approximately 30% of the total production expenditure. In the present study, a BNC-producing bacterium, Kosakonia radicincitans wava 1, was isolated from rotten Totapuri mango and evaluated for cost-effective BNC production using mango peel waste extract as a substitute for glucose in the conventional Hestrin–Schramm medium. Morphological analysis of the synthesized BNC using scanning electron microscopy revealed a dense, three-dimensional network of interwoven ribbon-like nanofibers with an average fiber width ranging from 88-111 nm. Process optimization was carried out using response surface methodology (RSM) with a randomized three levels factorial design, resulting in a quadratic model (p < 0.05). The optimal conditions for maximum BNC yield were determined to be 5% (w/v) mango peel extract concentration, pH 8.0, and an incubation temperature of 26 °C. Experimental validation under these optimized conditions produced a BNC yield (0.46 g/L) closely matching the model-predicted value (0.44 g/L), confirming the reliability of the statistical optimization. The results demonstrate that mango peel waste is an effective low-cost substrate for enhanced BNC production and offers a sustainable approach for valorizing agro-industrial waste.