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Soil micro-ecological imbalance is one of the critical factors associated with peach replanting issues. In an effort to understand the primary driving force in shaping soil microbial community, we analyzed soil nutrient, pH value, and autotoxin benzoic acid (BA) content, as well as rhizosphere microbial structure of peach seedlings grown in replanted soil (RS) and non-replanted soil (NS), both of which were collected from a same orchard. The results showed that depletion of soil nutrients, decreased soil pH, the accumulation of BA, and the deterioration of the soil microbial community were consistently detected in RS compared with NS, and led to the poor growth performance of peach seedlings. High-throughput sequencing analysis indicated that the soil microbial composition differed significantly between the NS and RS samples. Over time, the rhizosphere microbes of peach seedlings in RS and NS exhibited a similar trend of increasing bacterial richness and decreasing fungal richness. Interestingly, the NS had a high level of Bacillus and Ascobolus, whereas the RS enriched with Burkholderia and pathogenic Fusarium. Furthermore, canonical correspondence analysis revealed that soil nutrient and pH predominated the microbial composition of NS, whereas high level of soil BA played a crucial role in forming the microbial composition of RS. Altogether, this study provides a novel insight into better understanding of soil bacterial and fungal community structure in the RS, and aid in developing more effective means to alleviate the peach replant problem.