The aim of the study was to evaluate the microbiological and chemical contamination in settled dust at poultry farms. The scope of research included evaluating the contributions of the various granulometric fractions in settled dust samples, assessing microbial contamination using culture methods, concentrations of secondary metabolites in dust and their cytotoxicity against hepatocyte chicken cells by means of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) tests. In addition, we also evaluated the concentration of selected volatile odorous compounds (VOCs) using gas chromatographic and spectrophotometric methods and airborne dust concentration in the air with DustTrak™ DRX Aerosol Monitor. Studies were carried out on chicken broilers and laying hens at 13 poultry farms, with numbers of birds ranging from 8000 to 42,000. The airborne total dust concentration at poultry farms averaged 1.44 mg/m3 with a high percentage of the PM10 fraction (particulate matter with a diameter less than 10 μm). Microorganism concentrations in the settled dust were: 3.2 × 109 cfu/g for bacteria and 1.2 × 106 cfu/g for fungi. Potential pathogens (Enterococcus spp., Escherichia coli, Salmonella spp., Aspergillus fumigatus, Paecilomyces variotii) were also found. Secondary metabolites included aurofusarin, deoxynivalenol, 15-hydroxyculmorin zearalenone, zearalenone-sulfate, infectopyron, and neochinulin A. However, the dust samples showed weak cytotoxicity towards chicken hepatocyte cells, which ranged between 9.2% and 29.7%. Among volatile odorous compounds ammonia, acrolein, methyloamine, acetic acid, acetoaldehyde and formaldehyde were detected in the air. In conclusion, settled dust can be a carrier of microorganisms, odours and secondary metabolites in poultry farms, which can be harmful to workers’ health.