Monoclonal antibodies (IgM class) to Penicillium bilaii (isolate PB‐50) were developed and their specificity was determined against various fungi using enzyme‐linked immunosorbent assays. Cross‐reactivity in the range 0–6.5% was obtained for Fusarium, Aspergillus, Paecilomyces and Trichoderma species while 1.5–9.5% was obtained for P. lapidosum, P. crustosum, P. hiramayae, P. spinolosum and P. implicatum. Strains of P. bilaii (ATCC 22348 and ATCC 18309) yielded a relative cross‐reactivity of 15.9 and 10.8%, respectively. An immunochemical test for the detection of PB‐50 mycelia in plant and soil samples was devised using monoclonal antibodies. Several procedures have been proposed to show the feasibility of the application of developed monoclonal antibodies to P. bilaii PB‐50 detection in plant and soil samples. The immunodiffusion blotting method was superior to other tests for the detection of PB‐50 in infected plant samples.