Abstract: The utilization of herbal medicinal plants has gained significant attention for their therapeutic potential in disease treatment and health improvement. Ocimum sanctum, commonly known as Holy Basil or Tulsi, is a medicinal plant with a rich history in traditional medicine, particularly in Ayurveda. The Nanoemulsion was prepared using the ultra-sonication method, and its particle size, Polydispersity index (PDI), and zeta potential was characterized. The phytochemical profile of the Ocimum sanctum seed extract was determined, revealing the presence of flavonoids, phenols, terpenoids, alkaloids, saponins, and glycosides. The extractive yield was found to be 8.9%. The prepared Nanoemulsion exhibited a particle size of 100 nm, a low PDI of 0.245, and a zeta potential of -30 mV, indicating its stability. The in vitro MTT assay demonstrated concentration-dependent cytotoxicity of the Nanoemulsion on human cancer cell lines (HeLa), with higher selectivity towards cancer cells compared to normal cells (HEK-293). Furthermore, the Nanoemulsion showed concentration-dependent inhibition of COX-2, TNF-α, and IL-6 in an in vitro anti-inflammatory assay using peripheral blood mononuclear cells (PBMCs) treated with lipopolysaccharide (LPS). These findings highlight the potential therapeutic applications of the Ocimum sanctum seed Nanoemulsion in the treatment and management of diseases associated with inflammation.