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The current study was focused on the antimicrobial activities of Aspergillus flavus MTCC 13062 strain isolated from solid waste dumping site, Hisar (INDIA) which showed remarkable inhibitory effect against the test organisms (Streptococcus gordonii, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Pseudomonas florescens and Candida albicans). Response Surface Methodology (RSM) was performed to optimize the production of bioactive compounds by Aspergillus flavus MTCC 13062 which considerably increased the antimicrobial activity by 22%, 75% and 50% in E. coli, S. aureus and C. albicans, respectively at pH 7.5, temperature 28 °C and incubation period of 7 days. LC-MS was used to confirm the molecular weight and purity of the bioactive compound, while FTIRspectroscopy confirmed its structural characterization. Minimum inhibitory concentration (MIC) assay was carried out using dichloromethane extracts of Aspergillus flavus MTCC 13062 and results indicated MIC value as low as 25 μg/mL against S. aureus and E. coli while 50 μg/mL against C. albicans.Antifungal susceptibility test of Aspergillus flavus MTCC 13062 revealed that it was susceptible to Itraconazole, Fluconazole, Terbinafine, Kanamycin and Amphotericin B with zone of inhibition ranging from 10 to 24 mm. Furthermore, synergistic studies were carried out using commercial antibiotics in combination with the fungal extract and promising results were observed. We conclude that future implementation of the combinatorial results may help reduce the prescribed dosage of these antibiotics and thereby reducing the potential risks associated with the high doses of these harmful antibiotics.