Mangiferin (C19H18O11) is a C-glucoside xanthone that is mainly found in the leaves of mango (Mangifera indica L). The compound has been recognized for several pharmacological values, including antioxidant and antibacterial activities. Therefore, the present study aims to utilize the old leaves of mango as a potential source for mangiferin extraction and purification, and evaluate its antimicrobial and antioxidant properties. In the present study, mangiferin was extracted from a local variety of mango leaves using ultrasonic-assisted extraction methods and ethanol as the extraction solvent. The crude ethanolic extract of mangiferin was then purified by the liquid–liquid fractionation method with dichloromethane and ethyl acetate, then further separated by HPLC. The crude extract, ethyl acetate fractions and purified mangiferin were investigated for anti-microorganism activity against Escherichia coli, Salmonella spp. and Aspergillus flavus by using the paper disc diffusion assay. The results have shown that the extraction efficiency was 14.17%. The obtained mangiferin was 1.97 g of content and 94.2% of purity. The oxidation resistance of purified mangiferin was 1.77 times higher than the crude extract and 1.05 times higher than the standard mangiferin (IC50 = 13.841 μg/mL). However, the purified mangiferin has shown no inhibitory action against the experimental strains of microorganisms. The findings from the present study suggest an effective scheme of extraction and purification to obtain mangiferin from the local variety of mango with high purity and antioxidant potential.