<p>Gluconic acid is a mild organic acid produced by the dehydrogenation of glucose catalyzed by glucose oxidase. It has wide applications across food, pharmaceutical, chemical industries, etc. The growing demand has made it necessary for the isolation and characterization of potent organisms capable of enhanced production of gluconic acid using agro-industrial waste contributing to waste valorization. Therefore, an attempt was made to isolate, screen, and characterize the gluconic acid producing microorganisms from potato waste, a potential inexpensive substrate for bioconversion processes. Using the enrichment method, 15 fungal and 15 bacterial isolates were obtained. It was found that all the fungal and bacterial isolates showed positive for acid production when glucose (1%) was used as a carbon source, which is indicated by increased titrable acidity. Acid tolerance tests at different pH levels, such as 3, 4, and 5 showed that the isolates, especially fungi, exhibited greater tolerance at pH 5 and also showed slow growth at pH 3. Also, the statistical analysis using one-way ANOVA for the quantitative estimation of gluconic acid revealed that VDF 11 was producing the highest gluconic acid with 72.13 ± 1.82&#xa0;g/L. Further, the molecular characterization using NCBI BLAST_N of ITS ribosomal DNA and maximum likelihood method revealed VDF 11 as <i>Penicillium oxalicum.</i> Therefore, these outcomes highlight that the isolates can be utilized for the sustainable production of gluconic acid contributing to waste valorization.</p>