The importance of ethanol-based hand rubs (EBHRs) to prevent health care-associated infections is undisputed. However, there is a lack of meaningful data regarding the influence of EBHRs on skin microbiome.Four nurses in a neonatal intensive care unit were included. After a leave of 14 days, samples were taken before the first hand rubbing action and at the end of shift, with continued sampling on days 1, 7, and 28. To analyze the hand microbiome, microbial cells were collected using the glove-juice technique. Pro- and eukaryotic community profiles were created using amplicon sequencing of 16S and 18S ribosomal RNA (rRNA) gene markers.On average, hand antisepsis was performed 108 times per 8-hour work shift. Microbial communities were dominated by typical taxa found on human skin. In addition, a clear nurse-specific (ie, individual) microbiome signature could be observed. For Prokaryota, daily exposure led to the end-of-the-day microbiomes being more similar to each other across nurses. In contrast, longitudinal effect of 28-day application revealed more similarity of the Eukaryotic community.Frequent occupational use of EBHR did not adversely affect the composition of the human hand microbiome. Thus, daily hand antisepsis retains its significance as the most important procedure for infection control.