Species of the family Phytoseiidae (Acari: Mesostigmata) inhabit a wide range of herbs, shrubs, and trees. Horse chestnut, Aesculus hippocastanum, is an important ornamental tree in Europe and, in addition to its importance for pollinators, it can serve as a host plant of these predatory mites. Little is, however, known about the factors affecting spatiotemporal variability in the density of phytoseiids on A. hippocastanum in an urban environment. We therefore assessed the species composition and the spatial and seasonal variability in the abundance of Phytoseiidae species in the city of České Budějovice, South Bohemia, Czech Republic. Compound-leaf samples were randomly collected from horse chestnut tree branches at eight sites, five times during the vegetation season in 2013. The mites were collected by washing technique and mounted on slides for identification. In total, 13,903 specimens of phytoseiid mites were found, and eight species were identified: Amblyseius andersoni, Euseius finlandicus, Kampimodromus aberrans, Neoseiulella tiliarum, Phytoseius macropilis, Paraseiulus talbii, Paraseiulus triporus, and Typhlodromus (Typhlodromus) pyri. The predominant species was E. finlandicus (96.25%). The mean number of mites per compound leaf was 2.53, 10.40, 23.54, 11.59, and 9.27 on the sampling dates in each month between May and September, respectively. The results further revealed that the mite abundance varied significantly among sampling sites and that it was negatively related to percentage of greenery area, intensity of greenery care, distance to water body, and density and age of horse chestnut trees, while it was positively related to air pollution index. The importance of leaf micromorphology for the attractiveness of A. hippocastanum to Phytoseiidae is discussed.