Introduction: This study explores the optimization of growth conditions and the extraction, characterization and antimicrobial potential of chitosan derived from Rhizopus stolonifer, a filamentous fungus. R. stolonifer exhibited optimal biomass accumulation by the 5th day of cultivation (0.53g), after which growth plateaued; indicating the onset of the stationary phase. Maximum biomass was achieved at pH 6.0 and 30°C, with glucose as the most effective carbon sources, resulting to the highest fungal dry weight (0.483g). Methodology: Chitosan extraction was performed using modified method yielding maximum 81.4 mg on 7th day, with a calculated deacetylation degree of 72.53%, confirmed with FTIR spectroscopy through characteristic absorption bands at 1651 cm-1 and between 1032-1153cm-1. Results: The extracted chitosan showed concentration depending antimicrobial activity; showing strong inhibition effects against Bacillus cereus and Candida albicans (16mm at 1000 µg), moderate activities against Streptococcus mutans (11 mm) and limited or not activity against Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae. Conclusion: This result highlights suitability of R. stolonifer as an efficient and sustainable source of high quality chitosan, with promising use in agriculture, food preservation, also biomedicine specially for its selective microbial activity toward Gram-positive bacteria and fungi. © 2025 Elsevier B.V., All rights reserved.