The present study was initiated to analyze ticks collected periodically from the vegetation in an urban marshy biotope of central Europe. During the one-year-long study period, 1960 ticks were found, including Ixodes ricinus (n = 1037), Dermacentor reticulatus (n = 610) and Haemaphysalis concinna (n = 313). DNA was extracted from 199 Dermacentor reticulatus and 47 Ixodes ricinus, selected from the beginning of their questing period. Molecular analysis of the cytochrome c oxidase subunit I (cox1) barcoding gene with the classical Folmer primers revealed that among 37 D. reticulatus all except two ticks had serial mutations along a 129-bp-long part of the gene. The majority of these ticks were young, freshly molted adults. However, when the complete mitogenome was sequenced from two such "aberrant" ticks, these serial mutations were absent. In D. reticulatus, host DNA was detected from four synanthropic bird species, the dog, the red fox, the bank vole, the Eurasian shrew and the wild boar. Besides long-known endemic tick-borne pathogens, specimens of D. reticulatus and I. ricinus were shown to contain the DNA of Ehrlichia muris. Babesia microti had very high, 36% prevalence in I. ricinus. In conclusion, mutations in the cox1 fragment amplified with the Folmer primers were not present in the complete mitochondrial genome of D. reticulatus, indicating that probably nuclear mitochondrial DNA (NUMT) was amplified with the first method. To our knowledge, similarly high local prevalence of B. microti was only reported in ticks in North America, where this piroplasm is responsible for most cases of human babesiosis (unlike in Europe).