The aim of this study was to evaluate the effectiveness of ethyl alcohol (75%) disinfection of Japanese quail (Coturnix coturnix japonica) hatching eggs and analysis of microbial contamination of eggs during incubation, depending on their location in the setter. Disinfected eggshells were found to have lower total bacteria (TBC) and fungi (TFC) count. Concerning the vertical location of eggs (top, middle, bottom), disinfected eggs were characterized by similar values of the TBC (x = 1.54 log CFU/shell surface). For eggs without disinfection, it was found that those from middle and bottom levels of the setter had similar and lower TBC (by about 1.22 log CFU/shell surface) as compared to eggs from the top level. No statistically significant differences between levels were found in the case of TFC. Hatch breakouts (dead-in-shell embryos) from non-disinfected eggs were characterized by higher TBC (on average 0.37 log CFU/g). Disinfected eggs, located at the middle and bottom levels of the incubator, had similar and lower TBC in comparison with eggs from the top level. There were no microscopic fungi inside disinfected eggs of hatch breakouts. On the other hand, the non-disinfected eggs, placed on trays from the middle level of the incubator, had greater TFC (by about 0.9 log CFU/g) than those from top and bottom levels. Regardless of whether the eggs were disinfected or not, the largest group of microscopic fungi included Aspergillus and Penicillium.