Monoclonal antibodies (MAbs) were raised against Penicillium aurantiogriseum var. melanoconidium. Cross‐reactivity studies were carried out against 12 ‘field’ and 27 ‘storage’ fungi. Two MAbs (MAbs 32 and 37) reacted with all of the fungi tested and a third (MAb 38) with 38 out of the 39 fungi (although weakly with some). Monoclonal antibodies 32 and 37, but not MAb 38, were found to react to a degree with ‘clean’ barley. Enzyme‐linked immunosorbent assay (ELISA) using MAb 38 and extracts of barley inoculated with the homologous antigen showed a clear relationship between absorbance and amount of fungal growth. It is suggested that these MAbs could be used in a broad‐spectrum assay to detect fungi of significance to the quality of foods and feeds.