Microsporum canis NCPF 179 and M. canis NCPF 177 dermatophyte cytoplasmic extracts (DCEs) were used as antigens to generate monoclonal antibodies (mAbs). Hybridomas with supernatants of optical density (OD) > 1 for homologous dermatophyte cytoplasmic extracts (CE) and OD < 0.5 for heterologous CE of Candida albicans tested by the enzyme-linked immunosorbent assays (ELISA) were selected for cloning. mAbs secreted by cloned hybridoma lines were screened against CE of M. canis, M. gypseum, M. equinum, M. distortum, Trichophyton verrucosum, C. albicans, Malassezia pachydermatis and Aspergillus fumigatus. The ELISA performed on clone supernatants identified a variety of different activities between the dermatophyte and other fungal CEs. The selected mAbs were used for immunoblotting of native and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Immunoblots with some of the mAbs tested allowed the differentiation of strains belonging to different dermatophyte species and isolates belonging to the M. canis.