The two aims of this study were to obtain stable thyme-oil loaded nanoemulsions using the statistical design of experiment method (DOE) and to confirm their antimicrobial and disinfecting properties. Thyme oil was used as the oil phase, ECO Tween ® 80 acted as an emulsifier, and the rest of the formulation was deionized water. Ultrasonication was chosen as the method of obtaining the nanoemulsions. It was checked whether the input parameters (oil concentration, emulsifier concentration, amplitude, and sonication time) had a significant impact on the output parameters (nanoemulsion particle size, polydispersity index, viscosity, and stability over time). For the formulations selected on the basis of the statistical data analysis, the values of minimum inhibitory concentrations (MIC) and minimum biocidal concentrations (MBC/MFC) were determined in relation to 10 bacterial strains and 10 strains fungi (filamentous fungi, yeast-like fungi). The results obtained from the statistical analysis showed that the optimal concentration of the thyme oil in nanoemulsion should amount up to 2%. Biological studies proved that the obtained formulation had stronger antibacterial and antifungal activity compared to pure oil. Moreover, it was shown that the nanoemulsion caused the required for disinfectants reduction of > 5 log of bacterial strains ( S. aureus, P. aeruginosa) and > 4 log of fungal strains ( C. albicans) after 30 minutes.