Despite its frequent use in many religious institutions, the microbiological quality of holy water is clearly underinvestigated. We analyzed the microbial load of 54 holy water samples, repeatedly taken in five Roman Catholic churches in the greater area of Villingen-Schwenningen, Germany, by means of aerobic colony counting and Matrix-Assisted Laser Desorption/Ionization (MALDI) Biotyping of representative isolates. Over all samples, colony counting indicated an average aerobic microbial load of 5.85 ± 3.98 × 103 colony forming units (CFU) ml-1 (average ± standard error of the mean (SEM)). Urban churches showed significantly higher contaminations than rural churches, probably owing to a greater number of visitors. Out of 145 bacterial isolates, 63 (43%) were identified to genus level and 39 (27%) to species level. The majority of the identified bacteria were typical human skin commensals, mainly affiliated with the genus Staphylococcus. Ten out of 20 (50%) of the identified species were classified as potential pathogens. Appropriate hygiene measures should be taken to control microbial contamination of holy water, e.g., regular water exchange, particularly in highly frequented churches.