<p>Botulinum neurotoxins (BoNTs) are the most pernicious toxin and category ‘A’ bioterrorism agent, responsible for ‘botulism’, a rare but fatal disease. Toxins are divided into seven toxinotypes from A-G, where A, B, E, and F are accountable for human botulism. The available treatment strategies are inadequate and ineffective for post-neuronal intoxication. We report novel 2-[(8-hydroxyquinolin-7-yl)(phenyl)methylamino]benzoic acid derivatives targetingthe catalytic site of VAMP-acting serotypes (BoNT/B and F). Inhibitory potential of the synthesized compounds was studied using a framework of in silico and experimental approaches. Inhibitory activity and binding affinity were evaluated using substrate-based cleavage and SPR assays. Compound efficacy was tested in mice through pre-mixed, prophylactic, and therapeutic strategies. Molecular dynamics simulations analyzed binding interactions, structural fluctuations, and complex stability. Endopeptidase assay revealed that the selected compounds displayed ≥ 80% inhibition of BoNT/B and F catalytic activity, with IC₅₀ values ranging from 17.58 to 34.05 µM. SPR analysis displayed binding affinity of these molecules ranging from 8.13E-06 to 7.69E-04 for both the proteins. In the mouse bioassay, the selected molecules displayed complete protection and extension in survival of up to 20-fold. MD simulation study supported the experimental finding, revealing key interactions with HExxH and other active site residues forming stable conformation throughout the simulation time. Among them, compounds A15 and A36 were predicted to be more effective inhibitors of BoNT/B and F serotypes, respectively. These findings could lay a promising way for the development of novel therapeutics by reducing disease severity, enhancing survivability, and recovery where no post-exposure therapy presently available.</p>