The exposure to biocontaminants in animal facilities represents a risk for developing infectious, allergic and toxic diseases. The aim of this study was to determine what factors could be associated with a high level of exposure to biological agents through the measure and characterization of airborne fungi, bacteria, endotoxin, (1,3)-β-d-glucan and animal allergens. Airborne microorganisms were collected with an air sampler and identified by microscopic and biochemical methods. Endotoxin, (1,3)-β-d-glucan, Mus m 1, Rat n 1, Can f 1, Fel d 1, Equ c 4 allergens were detected on inhalable dust samples by Kinetic LAL, Glucatell, and ELISA assays, respectively. Our data evidenced that changing cages is a determinant factor in increasing the concentration of the airborne biocontaminants; the preparation of bedding and distribution of feed, performed in the storage area, is another critical working task in terms of exposure to endotoxins (210.7 EU/m3) and (1,3)-β-d-glucans (4.3 ng/m3). The highest concentration of Mus m 1 allergen (61.5 ng/m3) was observed in the dirty washing area. The detection of expositive peaks at risk of sensitization (>2 μg/g) by Fel d 1 in animal rooms shows passive transport by operators themselves, highlighting their role as vehicle between occupational and living environments.