Abstract: The antagonistic activity of 8 isolates of Pisolithus spp. associated with Quercus suberand Eucalyptus spp. was studied in vitro against 5 phytopathogenic fungi (Armillaria mellea,Botrytis cinerea, Curvularia lunata, Fusarium oxysporum and Helminthosporium maydis) inorder to select the isolates showing the highest antagonistic activity. These isolates showeddifferent level of antagonistic activity against the tested pathogens. Antagonism by directconfrontation was confirmed by using the liquid culture filtrate of the isolates. The molecularanalysis with ITS-RFLP restriction enzymes HinfI, MspI and TaqI, allowed the identification of asingle genotype for isolates of Pisolithus from eucalyptus and two genotypes from cork-oak.