Nine isolates of Trichoderma were collected from Assiut Governorate, Egypt, as leaf surface and endophytic fungi associated with onion flora stalks. Four isolates were identified as Trichoderma harzianum, while five isolates were belonging to Trichoderma longibrachiatum. The antagonistic activity of these isolates against onion purple blotch pathogen Alternaria porri was studied in vitro using dual culture assay. All tested Trichoderma isolates showed mycoparasitic activity and competitive capability against the mycelial growth of A. porri. Mycoparastic activity of Trichoderma was manifested morphologically by the overgrowth upon the mycelial growth of the pathogen and microscopically by production of coiling hyphae around pathogen hyphae. Isolates of T. harzianum exhibited high ability to compete on potato dextrose agar (PDA) medium causing the maximum rate of pathogen inhibition (73.12%), while isolates of T. longibrachiatum showed inhibition rate equalling 70.3%. Chitinase activity of Trichoderma was assayed, and T. harzianum Th-3013 showed the maximum value contributing 2.69 U/min. Application of T. harzianum Th-3013 to control purple blotch disease in vivo under greenhouse conditions caused disease reduction up to 52.3 and 79.9% before and after 48 h of pathogen inoculation, respectively, while the fungicide Ridomil Gold Plus caused disease reduction comprising 56.5 and 71.7%, respectively. This study proved that T. harzianum Th-3013 as a biocontrol agent showed significant reduction in onion purple blotch disease compared with the tested fungicide.