The present study aimed to investigate the antifungal bio-efficacy of four Trichoderma spp. (T. harzianum, T. hamatum, T. viridie and T. galaucum.) against Fusarium oxysporum f. sp. betae ( F. o. b.) which causing wilt disease of sugar beet . Screening the antifungal activities using dual and disc methods, showed that all the tested Trichoderma spp. recorded significant antagonistic effect against F.o.b.. Out of the four Trichoderma spp. T. harzianum and T. hamatum recorded the highest inhibitory effect on the mycelial growth of F.o.b. where the inhibition reached to 72.53 and 71.30 % and 83.50, 80.00 %respectively. After optimization of cultural conditions of both the selected Trichoderma spp., the inhibitory effect on F. o.b. were maximized to 85.50 and 82.20% respectively and more inhibitory effect reached to 86.70% was obtained with using the combination between the filtrates of both Trichoderma spp. . The identification of F. oxysporum f.sp betae, T. harzianum and T. hamatum were confirmed by molecular identification based on internal transcribed spacer (ITS) sequences of rDNA genes. The results showed that F. oxysporum f.sp betae exhibited similarity 99% to F. oxysporum f.sp. betae, with acc. no. PP410242, T. harzianum exhibited similarity % to T. harzianum with acc. no. PP410286 and T. hamatum exhibited similarity 100 % to T. asperellum with acc. no. PP410284. Extracts of T. harzianum and T. hamatum were analyzed using GC-MS to determine the active chemical constituents. The data showed a numerous compounds produced by the two Trichoderma spp. possessing high antimycotic property.