Melianone, the protolimonoid (24, 25-epoxyflindissone), was isolated from the medicinal tree species, Swietenia mahagoni (L.) JACQ (Meliaceae). The compound isolated from petroleum ether leaf extracts (5.39%) was quantified using high-performance thin-layer chromatography (HPTLC) method. In antimicrobial assays melianone inhibited Salmonella ser. Typhi with an MIC of 0.053 µM. Induced Fit Docking (IFD) of the ligand, melianone, with proteins involved in anaerobic virulence of the pathogen, revealed that it binds with FocA (a transport protein of formate ions) at its “periplasmic opening” with a glide energy of − 51.8576 kcal mol−1. Melianone altered the overall conformation of the protein (protomer A) by 0.347 Å RMSD. It induced a notable protein topology (Ω loop region) shift in the channel from an intermediate-open to a closed-state conformation and was supported by molecular dynamic simulations performed. FocA, a protein that contributes to its survival under anaerobic conditions, was further evaluated experimentally, after exposure of Salmonella ser. Typhi to melianone, resulting in the altered homeostasis of formate. © 2020, King Abdulaziz City for Science and Technology.