Staphylococcus aureus is an important Gram-positive pathogen responsible for a wide range of infections in humans and animals, including mastitis, skin infections, and systemic diseases. The increasing resistance of S. aureus to conventional antibiotics has encouraged the exploration of plant-based antimicrobial agents as potential alternatives. The present study evaluated the In-vitro antibacterial efficacy of aqueous leaf extract of Azadirachta indica against Staphylococcus aureus isolates obtained from small ruminants and their human handlers. Antibacterial activity of the neem extract was assessed using the agar well diffusion method, along with determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The neem extract demonstrated concentration-dependent antibacterial activity against S. aureus isolates, producing a maximum zone of inhibition of 15.5 mm at higher concentrations. Both MIC and MBC were recorded at 125 mg/mL, indicating a bactericidal effect of the extract. The findings suggest that Azadirachta indica possesses significant antibacterial activity against Staphylococcus aureus and may serve as a promising herbal alternative to conventional antibiotics. Further studies are required to isolate active phytochemicals and evaluate their therapeutic applicability.