Eucalyptus leaf and shoot blight fungal pathogens (Cryptospori-opsis eucalypti, Cylindrocladium sp. and Teratosphaeria destructans),isolated from symptom leaves and tested for pathogenicity, wereused for antagonistic assay. All actinomycete strains were screenedagainst C. eucalypti and Cylindrocladium sp. by the dual culture tech-nique and against T. destructans by the agar overlay technique.The results revealed that the degree of antifungal activity variedgreatly among the isolates. Comparison for antagonistic activitybetween endophytic actinomycetes and the isolates from the rhi-zosphere soil (Supplementary material Table A1) showed that thenumber of root endophytic actinomycetes with antagonistic activ-ity for controlling C. eucalypti (69.5%) was higher than those fromrhizospheric soils (53.9%). Activity against Cylindrocladium sp. wasthe least frequent with 15.8% from root endophytic strains, com-pared to 27.0% of those from rhizospheric soils. From a total of477 isolates, only 48 isolates (10.1%) displayed activity against allthree fungal pathogens. Among these, strain EUSKR2R82 showedthe highest activity by completely inhibiting the growth of all testedfungal pathogens. Two hundred and seventy two isolates (57.0%)inhibited C. eucalypti in which 17 isolates showed a maximum inhi-bition of 100%. One hundred and eighteen isolates (24.7%) inhibitedCylindrocladium sp. with 3 isolates exhibiting a maximum 100%of growth inhibition. A study of actinomycetes on spore germina-tion of T. destructans showed that 241 isolates (50.5%) could inhibitspore germination. Among these 477 isolates, 380 isolates (79.7%)inhibited at least one of these three serious pathogens while 96isolates (20.1%) did not show any antifungal activity.