Defensins and defensin-like peptides are functionally diverse commonly presented as an immune reaction between plant and pathogen . Trichoderma viride and Bacillus subtilis as biological control agents were inoculated into the soil to suppress the activity of the pathogenic Fusarium oxysporum and Rhizoctonia solani on roots of tomato (Solanum lycopersicum L.). The up- and down–regulated genes were examined in both biological control agent-treated plants and non-treated plants (control) using the differential display PCR. In treated plants, many up-regulated genes (21) with different molecular sizes ranging from 50 bp to 7 kbp were observed. Only four up-regulated genes were isolated from plants treated with B. subtilis +R. solani. The sequence and sequence analysis revealed that the identified genes were defensin genes; Amino Acid/Auxin Permease (AAAP) Family, Endopolygalacturonase PG1, Fructose-1,6-bisphosphatase and glycosyl transferase. Moreover, Chitinase gene, defensin genes (DF1 and DF2) were quantitatively determined using RT-PCR. The comparative expression level of the three induced genes was exponentially increased as a function of time after the application of the biological control agents. However, while the expression level of DF1 and DF2 was high in plants infected with either F. oxysporum or R. solani in the beginning of the experiment, the highest expression level of these genes was attained in the tomato plant treated with either T. viride or B. subtilis after 24 hour post inoculation.