We examined the selectivity of 53 sets of primers for environmental monitoring of indoor air quality. Thirty-six fungal strains, representing 26 species from 14 genera of commonly occurring fungi, and 16 different bacterial strains, representing both gram-negative and gram-positive species, were included in the experiment. We verified the specificity of 28 of the 53 sets of primers, which were classified as universal fungal, universal bacterial, group or species specific. The PCR conditions required for optimal specificity were also determined. These results can serve as a guide for the step-wise PCR-based detection and identification of airborne fungi commonly found in indoor environments.