Poplar anthracnose is one of the most serious diseases caused by the fungal pathogen Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. Biocontrol is an efficient green way for the disease control, and numerous researches have focused on exploring the potential biocontrol bacteria strains against C. gloeosporioides. In this study, antifungal activities against C. gloeosporioides of 108 rhizosphere soil isolates from healthy polar plants were investigated in vitro by the dual culture assay. The results suggested that strain ZSH-1 showed the highest level of antifungal activity, as it inhibited C. gloeosporioides at a distance of 10.00 mm. Based on the morphological, physiological-biochemical characteristics, and phylogeny analysis, strain ZSH-1 was identified as Bacillus subtilis. The sterile culture filtrate, crude protein, and crude lipopeptide extracts from the culture filtrate, and volatile compound(s) of ZSH-1 displayed a strong antagonism towards 7 fungal phytopathogens (C. gloeosporioides, Fusarium oxysporum, Alternaria tenuissima, Cytospora chrysosperma, Botryosphaeria dothidea, Mucor sp., and Absidia sp.), with inhibition rates ranging from 44.0 to 89.1%, 26.7 to 85.4%, 11.6 to 89.7%, and 7.8 to 63.2%, respectively. Moreover, ZSH-1 exhibited cell wall-degrading traits by producing 3 lytic enzymes (cellulose, β-1,3-glucanase, and protease). Finally, the greenhouse studies also revealed that strain ZSH-1 had a 47.6% (12 days) efficacy in controlling poplar anthracnose when compared with the control. In concluding, obtained results demonstrate the potential biocontrol effect of B. subtilis ZSH-1, and it can be used as a promising biocontrol agent against poplar anthracnose and other fungal phytopathogens.