Background: An aquatic macrophyte Monochoria hastata (L.) Solms has ethnomedicinal application in various ailments and experimentally is proved to have antibacterial property. Objectives: To evaluate the optimized extraction methods and solvents for antibacterial activity and their characterization by gas chromatography-mass spectrometry (GC-MS). Materials and Methods: Extractions from the aerial parts of the plant in solvents with different polarities and various techniques, viz., microwave, infusion, Soxhlet, and maceration, were used to evaluate the best antibacterial efficacy by agar well diffusion method. The phytochemical constituents present in the bioactive extract were analyzed using standard phytochemical screening methods and characterized by GC-MS analysis. Results: Ethyl acetate extract derived by the Soxhlet method showed the highest antibacterial activity against all the test bacterial strains, and Gram-positive strains were more susceptible than Gram-negative strains. The crude extracts showed antibacterial activity which ranged from 7.0 ± 0.3 to 16.5 ± 0.8 mm at 100 mg/ml. Fifty percent of methanol had the highest extractive value (21.3%) in the Soxhlet method. Phytochemical tests showed the presence of alkaloids, phenols, flavonoids, terpenoids, glycosides, and fats in the bioactive ethyl acetate extract. GC-MS analysis showed the presence of different fatty acids and their saturated esters as the principal components. The major compounds were tridecanoic acid, methyl ester, 2-hexyldecanoic acid, dodecanoic acid, and diethyl phthalate. Conclusion: The results suggest that the ethyl acetate extract of M. hastata (L.) Solms possesses some bioactive volatile compounds including fatty acids and their esters, which have antibacterial potentiality. This is the first report of such antibacterial assessment from M. hastata (L.) Solms.