It’s well known that synthetic antimicrobial drugs can cause adverse reactions such immunosuppression, toxicity, and hypersensitivity. The continued evolution of multi-resistant strains necessitates the continuous discovery of novel medications. The aforementioned adverse effects reduce the therapeutic efficacy of the currently prescribed antimicrobial drugs, demanding the search for alternative treatments. Due to its natural origins and negligible side effects, the field of herbal therapy has experienced exponential growth over the past several decades. The current goal of the work is to create an effective nanoemulsion of Moringa oleifera L. Seed Oil for antibacterial purposes. Titration technique was used to create the nanoemulsion, which was then evaluated for thermodynamic stability, pH, conductivity, viscosity, Refractive index, particle size distribution, and particle size analysis. Studies of the antimicrobial capabilities of gram positive and gram negative bacteria, as well as fungi, were conducted in vitro. According to ICH guidelines, nanoemulsion stability testing was conducted. Moringa seed oiltahen as oil phase, Tergitol 15-S-20 (surfactant), and transcutol P (co-surfactant) are used to formulate a nanoemulsion percentage composition of Oil:Smix(1:1):Water was 15:33:52. The following values were discovered to be 6.12± 0.124 for pH, 29.35 ±1.67 mP for viscosity, 1.404 ±0.024 for refrective index, and 10-4 s cm-1 for conductivity. The average particle size, polydispersity index, and zeta potential were determined to be 61.50 ± 0.512 nm, -28.4 mV±0.302, respectively. The stability study confirmed that the optimized nanoemulsion was sufficiently stable for3 months at room temperature. It was shown that the nanoemulsion of Moringa oleifera oil and Moringa seed oil had bactericidal and fungicidal properties with MICs of 0.110 g mL-1 and 0.123 g mL-1, respectively. As a result of the facts above, M. oleifera oil nanoemulsion could be a potential antimicrobial.