Thyme oil in water nanomulsion was prepared under subcritical water conditions using water and saponin, as solvent and emulsifier, respectively. Gas chromatography revealed that there were 44 bioactive components in the extracted thyme essential oil which, thymol and carvacrol were two mains of them. Experiments were designed based on central composite design and effects of amounts of saponin and thyme essential oil were evaluated on particle size, polydispersity index (PDI) and zeta potential of the prepared nanoemulsions using response surface methodology. Obtained results revealed that more desirable thyme oil nanoemulsions with minimum particle size (184.51 nm) and PDI (0.514), and maximum zeta potential (− 22.51 mV) were prepared using 0.94 g of saponin and 0.28 mL of thyme essential oil. Furthermore, results indicated that prepared nanoemulsion using obtained optimum production conditions had relatively high antioxidant activity (24%) and high antibacterial and antifungal activities against Staphylococcus aureus and Penicillium digitatum.