Heavy metals and antibiotics residues in agricultural soils are attracting more and more attention. A laboratory study was conducted to evaluate the single and combined effects of sulfadimidine (SM2) (0.05, 0.20, 0.80 mmol/kg) and copper (Cu) (1.60 mmol/kg) on soil microbial biomasses and ammoxidation microorganisms abundances after 7, 14, 21 and 28 days. The results demonstrated that the single and combined contaminations had a significant and persistent inhibitory effect on soil bacteria, fungi and actinomycetes populations and amoA gene copies of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) (Except SM2 0.05 and 0.20 mmol/kg on 7 and 14 d and SM2 0.05 mmol/kg on 21 d led to a stimulatory effect on fungi and AOA-amoA gene, respectively). With higher dosage and longer exposure time, the toxic effect of single and combined contaminants on soil bacteria, fungi and actinomycetes as well as on the amoA gene of AOA and AOB was greatly reinforced. Combined contaminants produced more toxicity than the chemicals were used alone. Overall, the interaction effects of SM2 and Cu on bacteria (on 14, 21 and 28 d), fungi and AOA-amoA were mainly synergism, in contrast, on actinomycetes (on 14, 21 and 28 d) and AOB-amoA were mainly antagonism. The order of the toxic effects of the single Cu and combined contaminants on microbial activity was: bacteria > actinomycetes > fungi. Furthermore, AOB-amoA was more sensitive to both contaminants toxicity than AOA-amoA, while AOA-amoA gene copies were greater than AOB-amoA gene copies about one order of magnitude.