Serine protease inhibitor (AMPI) was extracted from Aegle marmelos fruit pulp to explore its antimicrobial potential. Extraction was performed using a phosphate buffer with protease inhibitors, followed by ammonium sulfate precipitation and chromatographic purification using DEAE-cellulose ion-exchange and Sephadex G-75 and G-50 gel filtration columns. Biochemical characterization of AMPI showed that it retained inhibitory activity over a wide temperature range, with optimal performance at 30°C. Activity decreased sharply above 50°C, indicating thermal sensitivity beyond physiological limits. pH profiling revealed maximal stability between pH 6 and 8, with a notable decline under strongly acidic or alkaline conditions. AMPI maintained its function in the presence of non-ionic surfactants, but its activity was diminished by certain metal ions, particularly Fe³⁺ and Cu²⁺, suggesting potential oxidative vulnerability. AMPI was tested against S. aureus strains for its ability to inhibit and eradicate biofilms. At higher concentrations, the inhibitor demonstrated significant anti-biofilm effects, with MSSA showing the highest response. Biofilm eradication at 8× MIC levels exceeded 80% in certain strains, supporting AMPI's potential as a biofilm-targeting agent. Membrane permeabilization studies using standard fluorescence-based methods showed that AMPI induced gradual damage to both the outer and inner membranes in a time-dependent manner. To evaluate the regulatory impact of AMPI, sRNA expression profiling was conducted using quantitative real-time PCR. At 12 hours post treatment, early upregulation of RNAIII, Teg41, and Teg49 was observed in MSSA, possibly reflecting a transient stress response. By 24 hours, however, the expression of all tested sRNAs was markedly downregulated across MSSA, MRSA, and MDR-SA.