The long-term effects of combined divalent copper (Cu(II)) and tetracycline (TC) on the performance, microbial activity and community in a sequencing batch reactor (SBR) were investigated. The addition of Cu(II), TC or mixed Cu(II)/TC caused the decrease of the organics and nitrogen removal efficiencies, and their decreased degrees were the lowest at the addition of mixed Cu(II)/TC. The increase of mixed Cu(II)/TC concentrations in the influent did not change the antagonistic effects between Cu(II) and TC on nitrifying and denitrifying activities. Nitrifiers had higher tolerances to Cu(II), TC and mixed Cu(II)/TC than denitrifiers. Compared to the addition of Cu(II) or TC alone, the microbial community richness was higher at the addition of mixed Cu(II)/TC, while the microbial community diversity was lower. The increased protein (PN) in extracellular polymeric substances (EPS) was a protective response of bacteria to Cu(II), TC and mixed Cu(II)/TC.