Purpose The aim of the study was to evaluate exposure to moulds and house dust mite Dermatophagoides pteronyssinus in poultry farms, and related health effects in poultry workers (PW). Methods The study involved 41 PW and 45 control office workers. Working environment was evaluated for D. pteronyssinus allergen (Der p 1), moulds and endotoxin. In workers, eye, skin and respiratory symptoms, ventilatory lung function, atopy markers (skin prick test to inhalatory allergens, total IgE) and specific IgG to moulds were assessed. Results Der p 1 levels ranged <0.1–3.3 μg/g, exposure to fungi was 4.9 × 10 3 –6.8 × 10 4  cfu/m 3 , with prevailing Aspergillus , Penicillium and Mucor species, and endotoxin levels ranged 230–284 EU/m 3 . In comparison to control subjects, significantly higher prevalence of work-related nose, asthma, eye and skin symptoms, and slight decline in ventilatory lung function was found in PW. PW had significantly higher prevalence of IgG antibodies to moulds comparing to controls (63 vs. 36%, respectively, P  = 0.01), especially to Alternaria and Aspergillus species. The prevalence of atopy markers in PW was lower than in population-based studies. Conclusions Hazardous levels of Der p 1, endotoxin and moulds were determined in poultry houses. High prevalence of work-related symptoms and IgG antibodies to moulds was found in PW. Healthy worker effect is proposed as an explanation of low atopy markers prevalence among PW.