Colletotrichum nymphaeae is the principal causal agent of strawberry anthracnose worldwide, including Iran. For disease management, eco-friendly alternatives such as biological control instead of chemical fungicides are highly desirable. The aim of this study was to compare the efficacy of 20 strains of Trichoderma spp. indigenous to Iran against C. nymphaeae, to a commercial strain of Trichoderma atroviride (SC1) isolated elsewhere, under in vitro and in planta conditions. The tested strains belonged to T. harzianum (ten isolates), T. pleuroticola (five isolates), T. virens (three isolates), T. asperellum (one isolate) and T. afroharzianum (one isolate). In vitro results showed that most isolates used in this study were able to significantly inhibit mycelial growth of the pathogen in dual culture and non-volatile tests. The majority of the tested Trichoderma spp. isolates produced lytic enzymes, including chitinase, β-1,3-glucanase, protease and cellulase. Besides producing hydrogen cyanide, Trichoderma spp. strains were also shown to be positive in terms of siderophore production and the mycoparasitism process. In planta bioassays revealed that the best indigenous strain (T. virens CCTUT4) significantly reduced anthracnose, at the same rate as the commercial strain, although the standard chemical fungicide (Switch®) was superior in terms of disease control. A positive correlation between disease incidence and severity reduction was detected with increasing population density of the antagonists in this study. The findings of this study indicate that isolating Trichoderma-based biocontrol agents from the crop against whose disease are targeted does not necessarily provide advantages in comparison with a strain having good biocontrol properties and originating from a different substrate.