Wheat (Triticum aestivum L.) is an important crop cultivated successfully worldwide and in Egypt, which could be infected by Fusarium sp. Fungicides are generally used to control this pathogen. Nowadays, Trichoderma spp. and Pseudomonas fluorescens are used as biocontrol agents. In this study, a serological technique was carried out to explain the interaction between Trichoderma viride, P. fluorescens and the pathogenic fungus Fusarium semitectum using crossed-immunoelectrophoresis (CIE) and double diffusion reaction (DDR). In homologous reactions, the antigenic structures of F. semitectum, T. viride, and P. fluorescens were 7, 5 and 4 precipitin bands in CIE, while 2, 2, and 3 precipitin bands were detected in DDR, respectively. In heterologous reactions, antigens of T. viride electrophoresed against antibodies of F. semitectum gave two common bands between them in either of the used methods while P. fluorescens antigens gave only one common precipitin antigen when electrophoresed against antibodies of the pathogen using both methods. This clearly indicated that T. viride was able to recognize to the pathogenic fungus F. semitectum, which showed more common antigens.