Objectives: The main objective of the study was to determine the levels of house dust mite (Der p1), dog (Can f1), cat (Fel d1) and cockroach (Bla g2) allergens in kindergartens localized in an urban agglomeration. Material and Methods: A quantitative analysis of allergens was carried out in settled dust samples collected by vacuuming the floor surface in three kindergartens (N = 84) and children's clothing (N = 36). The samples were collected in springsummer and autumn-winter periods as well as at the beginning and end of the week. The allergen dust concentration was determined by enzyme-linked immunoenzymatic assay (ELISA). Results: The mean geometric concentrations (±geometric standard deviations) of allergens Der p1, Can f1, Fel d1 and Bla g2 determined in kindergartens were: 0.02±3.21 μg/g of dust; 0.97±4.49 μg/g of dust; 0.30±4.43 μg/g of dust and 0.01±3.08 μg/g of dust, respectively. Younger classrooms (children aged from 3 to 4 years) were characterized by almost twice higher mean concentration of allergen Fel d1, as compared to older classrooms (children aged from 5 to 6 years) (p < 0.05). A significant impact of seasonality on the level of dog allergen Can f1 was found (p < 0.05). No significant weekly variation was found in average concentrations of the allergens. Children who had a dog and/or cat at home were characterized by high concentrations of allergens Can f1 and Fel d1 on their clothes (59.2±5.39 μg Can f1/g of dust; 3.63±1.47 μg Fel d1/g of dust), significantly higher than concentrations of allergens in children who did not have any pets (p < 0.001). Conclusions: Special attention should be paid to keeping the kindergarten rooms tidy and clean and to an appropriate choice of furnishings and fittings which would prevent the proliferation of the house dust mite and accumulation of allergens.