The application of biochar stimulates the activities of microorganisms that affect soil quality and plant growth. However, studies on the impacts of biochar mainly focus on a monoculture, its effects on interspecific interactions are rarely reported. Here, we investigated the impacts of biochar on tomato/potato–onion intercropped (TO) in a pot experiment. Tomato monoculture (T) and TO were treated with no, 0.3, 0.6, and 1.2% biochar concentrations in a pot experiment. Microbial communities from tomato rhizosphere soil were analyzed by quantitative PCR and Illumina MiSeq. The results showed that compared with the tomato monoculture, 0.6%TO and 1.2%TO significantly increased tomato yield in 2018. TO and 1.2%TO significantly increased plant height and dry weight in 2018 and 2019. Biochar treatments increased soil pH, decreased NO3--N and bulk density, and increased the absorption of N, P, and K by tomato. Bacterial and fungal abundances increased with an increase in biochar concentration, while Bacillus spp. and Pseudomonas spp. abundances showed an “increase-decrease-increase” trend. Biochar had a little effect on bacterial diversities but significantly lowered fungal diversities. TO, 0.6%TO, and 1.2%TO increased the potentially beneficial organisms (e.g., Pseudeurotium and Solirubrobacter) and lowered the potentially pathogenic organisms (e.g., Kribbella and Ilyonectria). Different concentrations of biochar affected the bacterial and fungal community structures. Redundancy analysis indicated that the bacterial community was strongly correlated with soil pH, NO3--N, and EC, while the fungal community was closely related to soil NO3--N and moisture. The network analysis showed that biochar and intercropping affected the symbiosis pattern of the microorganisms and increased the proportion of positive interactions and nitrifying microorganisms (Nitrospirae) in the microbial community. Overall, our results indicated that monoculture and intercropping with biochar improved soil physicochemical states and plant nutrient absorption, and regulated soil microbial communities, these were the main factors to promote tomato growth and increase tomato productivity.