The pathogenesis-related proteins class 4 (PR-4) are known to be involved in plant defense response and/or related stress situations. The objective of this study was to evaluate the antifungal activity and reactive oxygen species (ROS) production of the TcPR-4b protein in Moniliophthora perniciosa. The TcPR-4b gene was cloned into pET28a and the resulting in frame fusion plasmid was used to transform Escherichia coli Roseta (DE3) for protein expression. The expression of the TcPR-4b recombinant protein was induced by 0.4 mM isopropyl-?-D-thio-galactoside and purified by immobilized metal affinity chromatography with TALON® Metal Affinity Resin. The TcPR-4b protein was used for in vitro assays against dikaryotic M. perniciosa broken hyphae. Then, 1 ml of the broken hyphae suspension was incubated for 2h with: i) 10 ?g of TcPR-4b in phosphate buffer (PB); ii) 20 ?g of TcPR-4b in PB; iii) 40 ?g of TcPR-4b in PB; iv) PB (control). Then, 1 ml of each treatment was applied on CPD solid medium (2% glucose, 2% peptone, 2% of agar) and incubated for 7 days at 25°C. The inhibition of hyphal growth was examined by counting the number of pseudo-colonies on three experimental replicates. To detect the production of the ROS in living cells of M. perniciosa, 1 ml of hyphae suspension was treated with 10 ?g of TcPR-4b in PB (or not - control) overnight at 25ºC, and then incubated at 25°C for 30 min with dihydroethidium which selectively stains the mitochondrial superoxide (O2 -). The hyphae were mounted on slides and observed under fluorescence microscope DMRA2 (Leica). Images were captured under fluorescent filters using the IM50 software (Leica). The reduction of M. perniciosa survival was observed in all tested concentrations of TcPR-4b with a decrease of survival correlated to the increase of the protein concentration. The hyphae treated with TcPR-4b presented a bright red fluorescence with specific more intense fluorescence in some foci. The control did not present fluorescenc