In recent years, extensive use of chemical fungicides can lead to negative effects on humans and environment, and can also launch resistance development in pathogens, so the biological control as an alternative or complementary strategies were studied. For the best use of these strategies, accurate and wide knowledge of the biology of pathogens and antagonists were needed. The goal of this research was to identify secondary metabolites which have produced by antagonists and investigating the correlation between them and the power of hindering the growth and pathogenicity of R. solani. Four selected isolates (N, H, A and Q) were studied. By the use of LOPAT analysis we determined that they were belonged to Pseudomonas fluorescens. Among them H had the most preventing activity against the fungi which was followed by N, A and Q, respectively. Q antagonist isolate had the most biofilm formation which was followed by H, A and N, respectively. H isolate had the most production of biosurfactant after that A, N and Q produced more lipopeptides, respectively. The results of analysis siderophore pyoverdine showed that H and N had the most production and the differences between them with Q and A were significant. Production of hydrogen cyanide was investigated. The results were interesting and showed that the weak isolates, A and Q had the most production of HCN but in N and H isolates were lower than them. The gene of producing HCN were studied by the use of AC primer and the presence of that was stablished. The comparison of producing Mupirocin antibiotic showed that H and Q had the most and A and N had the least ones. Using of Mup primer has indicated the presence of mupirocin producing gene. The potential of producing other antibiotics like 2,4-diacetylphlorogelocinol, pyoluteorin and phenazine were studied by the use of Phl, Plt and PCA primers frequently. The results showed that all isolates could synthesize these products. Analyzed bacteria had the destructive cell wall enzymes like cellulase, chitinase and protease. The microscopic pictures indicated that there is positive correlation between these enzymes with the biocontrol activity. The results of this research stablished the importance of secondary metabolites like enzymes, antibiotics, lipopeptides and pyoverdine in antagonistic property. The special role of each of these metabolites needs to be identified