Oregano essential oil (EO) has diverse biological activities. However, its instability and hydrophobic nature limit its incorporation in pharmaceutical preparations. The present study aimed to develop a stable oregano EO nanoemulsion (O_NE) and assess its antibacterial and anticancer effects compared to free (O_EO) and coarse emulsion (O_E) forms. Oregano EO was hydro-distilled and analysed via Gas chromatography-Mass spectroscopy and carvacrol (65.24%) and linalool (12.32%) were the major oil constituents. EO-based nanoemulsions were prepared using spontaneous nano-emulsification and ultrasonication at different time intervals. The nanoemulsion with a droplet size of 88.37 ± 1.004 nm (0.104 ± 0.002 PDI) exhibited the best stability profile. O_NE exhibited antibacterial activity against all the tested bacterial strains except Pseudomonas aeruginosa, and its activity was relatively higher against Staphylococcus epidermidis, Enterococcus faecalis and Escherichia coli than O_E. Nanoemulsion demonstrated a significant reduction of IC₅₀ values in A549 and MDA-MB-231 cell lines compared to O_E and free EO. Additionally, O_NE significantly inhibited colony formation and migratory abilities in both cell lines compared to O_E and O_EO. Moreover, a significant upregulation of the apoptotic markers CASP8 and BAX was demonstrated. A significant downregulation of antiapoptotic BCL2 and proliferation markers KI67 was also observed. To the best of our knowledge, this study is the first to scrutinize the anticancer effects of oregano oil nanoemulsion compared to conventional emulsion and free oil. Conclusively, the presently developed nanoemulsion is a promising nanoplatform for oregano EO as a potential antimicrobial and anticancer agent.