The distribution, fate, and effects of antibiotics and heavy metal residues in agricultural soil caused by long‐term application of organic fertilizers are of increasing concern. However, the ecotoxic effects of the interaction between antibiotics and heavy metals vary with the physicochemical properties of the soil, and it is still unclear how these substances interact with soil microbial functions. A short‐term microcosm experiment was conducted to investigate effects of the typical antibiotic oxytetracycline (OTC) with heavy metals (zinc [Zn] and copper [Cu]) alone or in combination on nitrification process and soil microbial activity in two different types of soil (FQ: sandy loam soil and NB: clay loamy soil). Results indicated that soil types influenced the toxic effects of antibiotics and heavy metals. Zn and Cu alone and when combined with OTC inhibited and retarded nitrification processes and reduced nitrous oxide emissions, which were mainly attributed to the inhibitory effects on ammonia‐oxidizing microorganisms. Moreover, Zn and Cu alone or combined with OTC increased soil respiration, but decreased the abundances of bacteria and fungi. ... mehrIn contrast, OTC alone had no significant effect on soil respiration but increased the abundance of fungi in both soils. Together, our results suggest that the widespread occurrence of antibiotics and heavy metals in agriculture soils may pose significant eco‐environmental risks by altering nitrification process and soil microbial activity.