<b><i>Background:</i></b> Exposure to microbes and their components may affect the maturation of the immune system.<b> </b>We examined the association of house dust microbial content with cytokine-producing capacity at birth and at the age of 1 year. <b><i>Methods:</i></b> Production of TNF-α, IFN-γ, IL-5, IL-8 and IL-10 at birth (n = 228) and at the age of 1 year (n = 200) following 24- and 48-hour whole-blood stimulation with staphylococcal enterotoxin B (SEB), lipopolysaccharide and the combination of phorbol ester and ionomycin was measured. Concentrations of ergosterol (marker for fungal biomass), muramic acid (marker for Gram-positive bacteria) and 3-hydroxy fatty acids with a carbon chain length from 10 to 14 (marker for Gram-negative bacteria) in living room floor dust were analyzed using gas chromatography-tandem mass spectrometry. Five single microbial species or groups were determined using a quantitative polymerase chain reaction method. <b><i>Results:</i></b> A high total level of the studied Gram-positive bacteria in general or <i>Mycobacterium </i>spp. in house dust was associated with decreased SEB-stimulated IFN-γ production, especially at the age of 1 year. The total level of indoor fungi analyzed <i>(Penicillium</i> spp<i>., Aspergillus </i>spp<i>. </i>and<i> Paecilomyces variotii</i> group, <i>Trichoderma viride/atroviride/koningii,</i><i>Wallemia sebi)</i> was also inversely associated with IFN-γ production at the age of 1 year, but this association did not remain significant after adjustment for potential confounders. A few associations were found between microbial exposures and other measured cytokines. <b><i>Conclusions:</i></b> High indoor microbial exposures may affect immune development in early life by reducing T helper type 1 cytokine secretion capacity. The observed hyporesponsiveness may reflect the adaptation of the immune system to environmental antigens. In future, more attention should be paid especially to the immunomodulatory role of exposures to Gram-positive bacteria.