Generally, rodents and other small mammals are considered as one of the sources of Toxoplasma gondii or Neospora caninum infection for cats and dogs as the definitive hosts of these two parasites, respectively. The aim of the study was to find out the prevalence of these two parasites in wild small mammals from the Czech Republic and to characterize T. gondii isolates by methods of molecular biology. A total of 621 wild small mammals were caught in the Czech Republic during years 2002 – 2014. Antibodies to T. gondii were detected by latex agglutination test in six (2.5 percent) of 240 small mammals (in two A. agrarius and four A. flavicollis). Antibodies to N. caninum were detected by commercially available competitive-inhibition enzyme-linked immunosorbent assay (percent inhibition value is more than 30 percent) in one A. flavicolis (0.4 percent). Three of 427 (0.7 percent) liver samples were positive for T. gondii by PCR while negative for N. caninum. All embryo samples (n = 102) were negative for both T. gondii and N. caninum. Three T. gondii isolates (two A. flavicollis and one A. sylvaticus) were genotyped by microsatellite markers and characterized as type II. To our knowledge, this is the first information about genetic characterization of T. gondii isolates in small mammals from Europe and the first detection of N. caninum antibodies in wild rodents from the Czech Republic.