Strawberry anthracnose disease, caused by Colletotrichum nymphaeae is a major limiting factor in strawberry production in Kurdistan province of Iran. In this research, endophytic fungi were isolated from stem and crown organs of strawberry and their antifungal activity against C. nymphaeae was tested. The selected isolates were identified as Penicillium hordei and Penicillium polonicum based on morphology and molecular characteristics. Antagonistic effects of selected isolates were evaluated against C. nymphaeae, through dual culture, volatile and non-volatile compounds production mechanisms in in vitro and greenhouse conditions. In volatile compounds test, two isolates of P. hordei and P. polonicum were able to reduce C. nymphaeae growth (34.05 and 42.36%) compared with control. In addition, isolates of P. hordei and P. polonicum reduced the strawberry anthracnose disease severity significantly in greenhouse conditions (58.9 to 61.4%) compared to the control. According to the results both in vitro and greenhouse experiments, both isolates reduced the growth of pathogen. Therefore, the results of this research showed that it may be possible to manage strawberry anthracnose disease effectively by using Penicillium species as biocontrol fungus. To our best knowledge, this is the first research related to the effects of Penicillium species on strawberry anthracnose.