Introducing an animal into a new location could be hazardous in the form of disease transmission, especially with respect to infections that are often overlooked. Such introduced infectious agents, including parasitic ones, then pose potential danger to the native animal population. Within the conservation program, the European bison was introduced into many European countries. However, this largest European herbivore was recognized as a new host for an invasive parasitic nematode, Ashworthius sidemi, in Poland in 1998. Since then, the prevalence of this non-native parasite in Poland has increased not only in bison but also in other wild ruminants. In 2011 five European bison individuals were transported from Poland to the Czech Republic. In the current study, we examined the gastrointestinal tracts of two European bison and two red deer culled in the Z. idlov game reserve. A. sidemi was identified in all investigated animals using both morphological and molecular methods; infection intensity was higher in bison than in deer. Our findings represent the first record of this invasive parasite in European bison at the Czech territory. The results of this study indicate changes in epidemiological patterns of Ashworthius infections in the climatic condition of Central Europe as well as the need to verify the reliability of ashworthiosis intravital diagnostics. One can expect A. sidemi to spread gradually in the Czech Republic and colonize other native ruminant hosts.