Ophidiomyces ophidiicola (Oo) is a snake fungal pathogen that causes ophidiomycosis. The disease manifests as dermatomycosis and/or systemic mycosis, and can be fatal. It occurs in free-ranging snakes in Asia, Europe and the USA and has also been demonstrated in captive snakes. We tested for the presence of Oo in free-ranging snake populations in the Czech and Slovak Republics (n = 420) between 2019 and 2022, focusing mainly on grass snakes (Natrix natrix) and dice snakes (Natrix tessellata), as well as various captive exotic species (n = 207). After collecting skin swabs, we tested for Oo using the qPCR method. We confirmed fragmented occurrence of Oo in the Czech Republic (total prevalence 15%) and recorded Oo in the Slovak Republic for the first time (total prevalence 33.9%). The highest prevalence was observed in N. tessellata (20.2%), which appears to be the most susceptible species. The pathogen was not detected in captive snakes.