This study focused on strawberry plant growth and the rhizosphere microbial community to analyze the effects of a new bio-organic fertilizer (BOF) which was prepared with Bacillus subtilis F2 as a functional strain. A strawberry pot experiment was conducted using the following three treatments: CK (control), organic fertilizer (OF), and bio-organic fertilizer (BOF). The agronomic traits, nutrient content and accumulation, enzyme activities in leaves, and rhizosphere microbial diversity of the strawberry plants were assessed. The application of BOF efficiently improved strawberry plant weight, promoted nutrient accumulation and content, particularly phosphorus (P) accumulation in shoots, remarkably accelerated malondialdehyde (MDA), and maintained peroxidase (POD) activities. Along with the application of BOF, the functional strain F2 existed and multiplied in the strawberry rhizosphere. Terminal-restriction fragment length polymorphism (T-RFLP) results showed that the application of BOF did not change the alpha-diversity and abundance of microbial communities, and even markedly decreased homogeneity. However, it did alter beta-diversity, as the largest interaction principle component axis (IPCA1) of additive main effect multiplicative interaction (AMMI) analysis revealed that 99.17% of variation in the microbial community was attributable to BOF application. The results demonstrated that this new BOF can significantly promote the growth of strawberry plants and alter microbial community structure, and these effects are probably attributable to the strain F2 or the comprehensive benefits of OF and F2, which were generated during the secondary composting of BOF, rather than to OF component itself.