Antibiotic resistance microorganisms (ARMs), especially gram-negative bacteria, are a global health threat. The impact of fermentation on phytochemicals is numerous, and exploring this potential is the front line of drug development. The study investigates the role of fermentation in modifying V. amygdalina leaf secondary metabolites as a potent antibiotic against Escherichia. coli, Bacillus subtilis, and Salmonella typhi. The results of this work showed that the fermentation process increased the content of lycopene, flavonoid, and carotenoid compounds but decreased chlorophyll, soluble protein, and phenol. Pearson's correlation heatmap showed a strong correlation between microbial activities and secondary metabolic changes. The methanolic extract of fermented V. amygdalina leaf pulp (VALP) showed significant antioxidant and anti-inflammatory activities. The GCMS and FTIR outputs showed unique compounds and structural modifications at different intervals of the fermentation period. In-silico analyses showed that 4,6-Cholestadien-3β-ol – a compound that is unique to the 9th day of fermentation was responsible for the inhibition of the gram-negative bacteria via multiple non-ionic interactions with key catalytic site residues and higher binding affinity to OmpC, OmpF, and RNase P than ampicillin. Therefore, this steroid-derived compound may open a new pipeline into the development of multi-target antibiotics against a broad-spectrum multidrug-resistant gram-negative bacterial.