The coexistence of antibiotics with sulfide and nitrate is common in sewage. Thus, this study explored the removal performance of nitrate and sulfide, and the response of extracellular polymer substances (EPS) and the microbial community to the sulfide autotrophic denitrification (SAD) process under oxytetracycline (OTC) stress. In Phase Ⅰ, the SAD system showed favouranle performance (nitrate removal rate ＞ 92.57%, sulfide removal rate ＞ 97.75%). However, in Phase Ⅳ, at OTC concentrations of 10, 15, and 20 mg/L, the NRE decreased to 76.13%, 40.71%, 11.37%, respectively, and the SRE decreased to 97.58%, 97.09%, 92.84%, respectively. At OTC concentrations of 0, 10, 15, and 20 mg/L, the EPS content were 1.62, 1.75, 2.03, and 1.42 mg/gVSS, respectively. The results showed that SAD performance gradually deteriorated under OTC stress. In particular, when the OTC concentration was 20 mg/L, the EPS content was lower than that of the control test, which could be attributed to the occurrence of microbial death. Finally, high-throughput sequencing results showed that OTC exposure led to gradual domination by heterotrophic denitrifying bacteria.