AbstractGarlic oil in water nanoemulsion was resulted through subcritical water method (temperature of 120 °C and pressure of 1.5 bar, for 2 h), using aponin, as emulsifier. Based on the prepared garlic oil nanoemulsion, astaxanthin–garlic oil nanoemulsions were prepared using spontaneous microemulsification technique. Response surface methodology was employed to evaluate the effects of independent variables namely, amount of garlic oil nanoemulsion (1–9 mL) and amount of provided astaxanthin powder (1–9 g) on particle size and polydispersity index (PDI) of the resulted nanoemulsions. Results of optimization indicated that well dispersed and spherical nanodroplets were formed in the nanoemulsions with minimum particle size (76 nm) and polydispersity index (PDI, 0.358) and maximum zeta potential value (−8.01 mV), using garlic oil nanoemulsion amount of 8.27 mL and 4.15 g of astaxanthin powder. Strong antioxidant activity (>100%) of the prepared astaxanthin–garlic oil nanoemulsion, using obtained optimum amounts of the components, could be related to the highest antioxidant activity of the colloidal astaxanthin (>100%) as compared to that of the garlic oil nanoemulsion (16.4%). However, higher bactericidal activity of the resulted nanoemulsion against Escherichia coli and Staphylococcus aureus, were related to the main sulfur bioactive components of the garlic oil in which their main functional groups were detected by Fourier transform-infrared spectroscopy.