Lemongrass (Cymbopogon flexuosus) essential oil (LGEO) contains α-citral, β-citral and other phytochemicals extracted using various methods. This research extracted essential oils using steam distillation (SD) and microwave-assisted hydro distillation (MAHD) to maximize quantity and purity. LGEO was tested for antibacterial properties. LGEO was extracted using SD and compared to MAHD output based on oil production and chemical composition. We performed GCMS to characterize LGEO. Fourier transform infrared spectroscopy (FTIR) used for quantum chemical analysis. Spectroscopic analysis showed that SD extracted secondary metabolites (ethyl-linalool, isogeranial, β-citral, α-citral, geranyl acetate, and caryophyllene) yielded 9.7 %, 11.5 %, 35.4 %, 13.4 %, 6.4 %, and 6.4 %, respectively, while MAHD yielded 10.2 %, 13.4 %, 43.2 %, 17.3 %, 6.9 %, and 7.3 %. MAHD extracted α and β citral content was better than SD extraction technique. FTIR spectroscopy and quantum chemistry analysis showed extracted oil chemical composition, electronic structure of α and β citral isomers. In the disc-diffusion experiment, both extracts were effective against Gram-positive and Gram-negative bacteria and harmful fungi. LGEO from SD and MAHD extraction (30 mg/mL) demonstrated disc diffusion assay antibacterial efficacy against microorganisms. The two extracts effectively inhibited microorganisms with MIC values of 3.75 and 7.5 μg/mL. It can be concluded that, LGEO have greater antimicrobial activity in MAHD extraction.