Changes in soil biological properties have been implicated as one of the causes of soil sickness, a phenomenon that occurs in continuous monocropping systems. However, the causes for these changes are not yet clear. The aim of this work was to elucidate the role of p-hydroxybenzoic acid (PHBA), an autotoxin of cucumber (Cucumis sativus L.), in changing soil microbial communities. p-Hydroxybenzoic acid was applied to soil every other day for 10 days in cucumber pot assays. Then, the structures and sizes of bacterial and fungal communities, dehydrogenase activity, and microbial carbon biomass (MCB) were assessed in the rhizosphere soil. Structures and sizes of rhizosphere bacterial and fungal communities were analyzed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and real-time PCR, respectively. p-Hydroxybenzoic acid inhibited cucumber seedling growth and stimulated rhizosphere dehydrogenase activity, MBC content, and bacterial and fungal community sizes. Rhizosphere bacterial and fungal communities responded differently to exogenously applied PHBA. The PHBA decreased the Shannon-Wiener index for the rhizosphere bacterial community but increased that for the rhizosphere fungal community. In addition, the response of the rhizosphere fungal community structure to PHBA acid was concentration dependent, but was not for the rhizosphere bacterial community structure. Our results indicate that PHBA plays a significant role in the chemical interactions between cucumber and soil microorganisms and could account for the changes in soil microbial communities in the continuously monocropped cucumber system.