In this study, inactivation’s of some fungal and bacterial species by Ultraviolet (UV) light were studied under different time and different surface conditions. We also aimed to determine surface-time relation in the inactivation of tested microorganisms. The fungi/-bactericidal and-static effect of UV light at different wavelengths was tested at different time periods of 1-75 min as well as on different surfaces such as microscope slide, aluminum foil, coin and solid medium. The distance between the UV light and the sample was 30 cm. It is the distance widely used in dental and cosmetic fields in commercial available boxes for sterilization of the tools (e.g., scissors, forceps, spatula etc.). It is found that exposure to UV light (254 nm) for 45 min was enough for inactivation of yeast like fungi and bacteria. Under the same conditions, fungicidal effects against molds were observed in 75 min in all surfaces except for the medium surface. In conclusion, different surface conditions and exposure time are important in UV light sterilization of microorganisms.