Strawberry root rot caused by Fusarium solani is one of the main diseases of strawberries and significantly impacts the yield and quality of strawberry fruit. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional chemical fungicides. To obtain antagonistic bacteria with a high biocontrol effect on strawberry root rot, over 72 rhizosphere bacteria were isolated from the strawberry rhizosphere soil and screened for their antifungal activity against F. solani by dual culture assay. Among them, strains CMS5 and CMR12 showed the strongest inhibitory activity against F. solani (inhibition rate 57.78% and 65.93%, respectively) and exhibited broad-spectrum antifungal activity. According to the phylogenetic tree based on 16S rDNA and gyrB genes, CMS5 and CMR12 were identified as Bacillus amyloliquefaciens. Lipopeptide genes involved in surfactin, iturin, and fengycin biosynthesis were detected in the DNA genomes of CMS5 and CMR12 by PCR amplification. The genes related to the three major lipopeptide metabolites existed in the DNA genome of strains CMS5 and CMR12, and the lipopeptides could inhibit the mycelial growth of F. solani and resulted in distorted hyphae. The inhibitory rates of lipopeptides of CMS5 and CMR12 on the spore germination of F. solani were 61.00% and 42.67%, respectively. The plant-growth-promoting (PGP) traits in vitro screening showed that CMS5 and CMR12 have the ability to fix nitrogen and secreted indoleacetic acid (IAA). In the potting test, the control efficiency of CMS5, CMR12 and CMS5+CMR12 against strawberry root rot were 65.3%, 67.94% and 88.00%, respectively. Furthermore, CMS5 and CMR12 enhanced the resistance of strawberry to F. solani by increasing the activities of defense enzymes MDA, CAT and SOD. Moreover, CMS5 and CMR12 significantly promoted the growth of strawberry seedlings such as root length, seedling length and seedling fresh weight. This study revealed that B. amyloliquefaciens CMS5 and CMR12 have high potential to be used as biocontrol agents to control strawberry root rot.