Microbial toxins and eukaryotic cell toxicity from indoor building materials heavily colonized by fungi and bacteria were analyzed. The dominant colonizers at water-damaged sites of the building were Stachybotrys chartarum (10 3 to 10 5 visible conidia cm ؊2 ), Penicillium and Aspergillus species (10 4 CFU mg ؊1 ), gram-negative bacteria (10 4 CFU mg ؊1 ), and mycobacteria (10 3 CFU mg ؊1 ). The mycobacterial isolates were most similar to M. komossense, with 98% similarity of the complete 16S rDNA sequence. Limulus assay of water extracts prepared from a water-damaged gypsum liner revealed high contents of gram-negative endotoxin (17 ng mg ؊1 of E. coli lipopolysaccharide equivalents) and ␤-D-glucan (210 ng mg ؊1 of curdlan equivalents). High-performance liquid chromatography analysis of the methanol extracts showed that the water-damaged gypsum liner also contained satratoxin (17 ng mg ؊1 ). This methanol-extracted substance was 200 times more toxic to rabbit skin and fetus feline lung cells than extract of gypsum liner sampled from a non-water-damaged site. The same extract contained toxin(s) that paralyzed the motility of boar spermatozoa at extremely low concentrations; the 50% effective concentration was 0.3 g of dry solids per ml. This toxicity was not explainable by the amount of bacterial endotoxin, ␤-D-glucan, or satratoxin present in the same extract. The novel in vitro toxicity test that utilized boar spermatozoa as described in this article is convenient to perform and reproducible and was a useful tool for detecting toxins of microbial origin toward eukaryotic cells not detectable in building materials by the other methods.