Central India is one of the largest biodiversity regions in Asia, characterized by a great range of medicinal and aromatic plants, whose therapeutic use reflect a longstanding heritage across several cultures. The genus Cymbopogon possesses significant medicinal characteristics and is recognized for its remarkable potential, including its use in cancer treatment in certain regions, thereby offering an alternative to allopathic therapies with natural remedies. This study aims to evaluate the antibacterial efficacy of the chemical and identify its specific constituents. The antimicrobial profile of hydro-alcoholic, aqueous, methanol, ethanol, hexane, chloroform, and petroleum ether extracts of Cymbopogon flexuosus demonstrated that the methanol and ethanol extracts exhibited significant antibacterial efficacy against pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Brevibacillus brevis, Enterococcus faecalis, Vibrio cholerae, Aspergillus niger, Candida albicans, Schizosaccharomyces sp., and Saccharomyces cerevisiae. The petroleum ether extract of Cymbopogon flexuosus demonstrated minimal efficacy against the test pathogens. FTIR and HPLC analyses have shown Quercetin as the predominant bioactive metabolite.