Antimony (Sb) primarily exists in trivalent (III) and pentavalent (V) speciation in the soil environment and poses a potential threat to ecological soil function as a toxic metalloid element. To evaluate the ecological effect of Sb in soil, the effects of different concentrations of Sb(III) and Sb(V) on microbial biomass carbon (MBC), soil basal respiration (SBR), potential nitrification rate (PNR), five enzyme activities and bacterial community structure were investigated using biochemical methods and high-throughput sequencing technology during the 1st and 8th weeks of exposure. The results of these experiments indicated that MBC and SBR were influenced, and PNR, FDA hydrolysis activity and urease activity were significantly inhibited by Sb(III) and Sb(V), while the activities of dehydrogenase, acid phosphatase and β-glucosidase had no clear effects on the amounts of Sb(III) or Sb(V). Meanwhile, there are some discrepancies regarding the effects of Sb(III) and Sb(V) on the same microbial indicators, and incubation time is not a neglected factor. Sequencing analysis revealed that Sb(III) decreased the bacterial diversity indexes and abundances of specific bacteria at the phylum level, whereas Sb(V) had little effect on them. The bacterial community structure at the genus level was altered by Sb(III) and Sb(V), in which the abundances of some functional microbes were increased, while the abundances of some functional microbes were reduced under Sb pollution. In general, PNR, FDA hydrolysis activity, urease activity and the abundances of specific functional microbes (i.e., Nitrospira) could be considered as sensitive indicators of Sb contamination. This study highlights the ecological effect of Sb(III) and Sb(V) on agricultural soil and will provides references for environmental monitoring and assessment.