The aim of this study was to assess the exposure of library workers to biological agents based on quantitative and qualitative characteristics of airborne and settled dust microflora supplemented with the analysis of dust mite allergens. The bioaerosol sampling was carried out using a 6-stage Andersen impactor. The settled dust samples were collected from book covers using cotton swabs and vacuum cleaner. Isolated microbial colonies were identified to the genus and/or species level. Moreover, the concentration of guanine as a predictor of dust mite allergen content was determined with the semi-quantitative Acarex test. The bioaerosol concentrations were low and they did not exceed the proposed Polish reference limits. The presence of air-conditioning or ventilating system resulted in the decreased biological contamination in libraries. The identification ofmicroorganisms in bioaerosol and settled dust samples revealed the presence of strains classified into group 2 according to their risk of infection. The level of dust mite allergens was elevated. Inhalation exposure to molds and dust mite allergens may result in the occurrence of allergic reactions and SBS symptoms.