Sansevieria trifasciata Prain holds great potential as a valuable asset in pharmaceutical development. In this study, our focus is to explore and assess the antibacterial activity of various components derived from this plant, including extracts, fractions, subfractions, and isolates, explicitly targeting two common bacteria: Escherichia coli and Streptococcus aureus. The isolated compound, identified as a derivative pyridone alkaloid (5-methyl-11-(2-oxopyridin-1(2H)-yl)undecaneperoxoicacid), demonstrates notable antibacterial effects. The extracts, fractions, subfractions, and isolates reveal significant bacterial growth reductions (p < 0.05). The minimum inhibitory concentration (MIC) values for Escherichia coli were 1.95 ppm, 3.9 ppm, 15.62 ppm, and 7.81 ppm, respectively, while the MIC values for Streptococcus aureus were 1.95 ppm, 1.95 ppm, 15.62 ppm, and 7.81 ppm, respectively. Computational analysis showed the isolates' interaction with key residues on the active site of beta-ketoacyl-ACP synthase from Escherichia coli and TyrRS from Streptococcus aureus. The findings indicate that the isolates exhibit a strong affinity for specific residues, including His333, Cys163, and Phe392 in beta-ketoacyl-ACP synthase, as well as Arg88, His117, Glu160, and Gln213 in TyrRS. Comparative energy calculations using MMPBSA demonstrate the isolates' favorable binding energy (-104,101 kJ/mol for beta-ketoacyl-ACP synthase and -81,060 kJ/mol for TyrRS) compared to ciprofloxacin. The elucidated antibacterial activity and molecular interactions of the isolates present valuable knowledge for future in vitro studies, facilitating the development of novel antibacterial agents targeting diverse bacterial strains.