Mold contamination and exposure to fungi in indoor environments has been associated with various adverse health effects but little is known about the significance of individual fungal species in the initiation or exacerbation of such effects. Using Stachybotrys chartarum as a model fungus we sought to demonstrate that monoclonal antibodies (mAbs) can provide species-specific diagnostic reagents and also be used to investigate immunological cross-reactivity patterns among fungi. Mice were immunized with S. chartarum spore walls and monoclonal antibodies were screened against 60 fungal species and 24 different isolates of S. chartarum using an indirect ELISA. One species-specific mAb (IgG(1)) reacted only with spore preparations but not mycelium of S. chartarum or propagules of any other fungus. Five cross-reactive mAbs (IgM) documented extensive cross-reactivity among nine related Stachybotrys species and several non-related genera including several species of Cladosporium, Memnoniella, Myrothecium and Trichoderma. We also found that the ELISA reactivity for cross-reactive antigens and different isolates of S. chartarum differed considerably for normalized total amounts of mycelial antigen. We demonstrate that mAbs and immunoassays have the potential to detect S. chartarum species-specifically. The observed reactivity patterns with cross-reactive mAbs suggest that several fungi may share common antigens and that the majority of antigens are expressed by spores and mycelia. The observed cross-reactivity patterns need to be considered for accurate interpretations of environmental and serological analyses.