The partial substitution of chemical fertilizers with organic manure has positive effects on crop productivity and sustainable development. Nevertheless, few studies have focused on major grain crops. Herein, we report the short-term effects of the partial substitution of chemical fertilizers with organic manure on the physicochemical properties, microbial community, and enzyme activities in the rhizosphere soil of a maize (Zea mays L.) field. A decrease in soil bulk density, pH, and electrical conductivity, concomitant with an increase in soil urease, invertase, and alkaline phosphatase activities, and a high level of nutrients were observed in organic manure-treated soil. The influence of the organic substitution treatment on bacterial diversity was greater than that on fungal diversity, particularly on alpha diversity. Among dominant bacterial phyla, Actinobacteria abundance changed the most, with significantly increase under organic manure treatment. In turn, among fungi, only Ascomycota responded substantially to organic substitution. Binding spatial ordination analysis revealed that relative soil water content and soil organic carbon, and nitrate and total nitrogen contents had a stronger effect on bacteria and fungi, respectively, than any other soil physicochemical property. Additionally, the changes in bacterial and fungal communities influenced soil enzymatic activities. Moreover, partial least squares path model revealed that soil physicochemical properties indirectly affected soil enzymatic activities by their direct effects on microbial (both bacteria and fungi) community. Overall, our results indicate that the substitution of chemical fertilizers by organic manure changed the composition of the soil microbial community, and that the effects of the substitution were more significant on bacteria than on fungi.