Peanut brown root rot is a rhizoplane disease caused by the soil-borne pathogen Fusarium solani. The objective of this study was to determine the genetic and physiological mechanisms of T. harzianum ITEM 3636 involved in the antagonism against the phytopathogenic fungi F. solani, and to evaluate its biocontrol effect on peanut brown root rot in greenhouse assays. The in vitro tests showed that T. harzianum ITEM 3636 exert its antagonistic activity against F. solani RC386 through the synthesis of secondary metabolites, high enzymatic activity (chitinases 0.054 U ml- 1, N-Acetyl-beta-D-glucosaminidases 0.21 U ml-1, proteases 0.063 U ml-1 and glucanases 0.139 U ml-1) and important modifications in the pathogen hyphae. In the gene expression analysis of biocontrolassociated genes (prb1, chit33, bgn13.1) an upregulation was detected when T. harzianum ITEM 3636 interacted with F. solani RC386 mycelia. The greenhouse assays showed that the previous application of T. harzianum ITEM 3636 on peanut seeds generated a protective effect in peanut plants which were then affected by F. solani, since it reduced both the incidence and the severity of peanut brown root rot, by 3.8% and 63.98% respectively. In conclusion, T. harzianum ITEM 3636 strain could be considered as a biofungicide against F. solani in microbial formulations intended for peanut plants.