Strawberry root rot caused by Neopestalotiopsis clavispora is one of the main diseases of strawberries and significantly impacts the yield and quality of strawberry fruit. Currently, the only accessible control methods are fungicide sprays, which could have an adverse effect on the consumers of the strawberries. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional synthetic chemical fungicides. Trichoderma spp. are frequently used as biological agents to prevent root rot in strawberries. In order to provide highly effective biocontrol resources for controlling strawberry root rot caused by Neopestalotiopsis clavispora, the biocontrol mechanism, the control effects of T. asperellum CMT10 against strawberry root rot, and the growth-promoting effects on strawberry seedlings were investigated using plate culture, microscopy observation, and root drenching methods. The results showed that CMT10 had obvious competitive, antimycotic, and hyperparasitic effects on N. clavispora CMGF3. The CMT10 could quickly occupy nutritional space, and the inhibition rate of CMT10 against CMGF3 was 65.49% 7 d after co-culture. The inhibition rates of volatile metabolites and fermentation metabolites produced by CMT10 were 79.67% and 69.84% against CMGF3, respectively. The mycelium of CMT10 can act as a hyperparasite by contacting, winding, and penetrating the hyphae of CMGF3. Pot experiment showed that the biocontrol efficiency of CMT10 on strawberry root rot caused by Neopestalotiopsis clavispora was 63.09%. CMT10 promoted strawberry growth, plant height, root length, total fresh weight, root fresh weight, stem fresh weight, and root dry weight by 20.09%, 22.39%, 87.11%, 101.58%, 79.82%, and 72.33%, respectively. Overall, this study showed the ability of T. asperellum CMT10 to control strawberry root rot and its potential to be developed as a novel biocontrol agent to replace chemical fungicides for eco-friendly and sustainable agriculture.