The overuse of antibiotics in the medical and aquaculture industries has led to the frequent detection of antibiotics in wastewater. Considering antibiotics would have an unknown impact on wastewater treatment in the future, the long-term effects of sulfamethoxazole (SMX) and tetracycline (TC) stress on the performance, functional genes and microbial community in three bioretention cells were investigated. The results showed that during the experiment, 0.8–1.2 mg/L of SMX would not destroy the water treatment capacity of the bioretention cells, and had a promoting effect on total nitrogen and ammonia nitrogen. 1.6 mg/L of SMX would cause the reduction of nitrogen removal efficiency and the phenomenon of phosphorus release, but it could be restored after a period of operation. TC of 0.8–1.2 mg/L did not have a significant impact on the removal of nutrients in AC-BRC (activated carbon-bioretention cell) and ACI-BRC (activated carbon and iron-bioretention cell), but TC of 1.2 mg/L caused the phenomenon of phosphorus release in BRC and the decrease of total nitrogen removal rate, 1.6 mg/L TC could make the bioretention cell lose its water treatment capacity. qPCR analysis of denitrification genes showed that the abundance of nirS, nirK, nosZ, and hzo had varying degrees of decrease before and after antibiotic stress, which meant the two antibiotics significantly inhibited the reduction of nitrite and nitrous oxide. But for the total number of bacteria, the relative abundance of the four genes has increased. The results of microbial community analysis also found that Proteobacteria, Bacteroidetes, Chloroflexi, and BIrii41, Denitratisoma, Ferritrophicum, Thiobacillus occupied the dominant species at the phylum level and the genus level respectively, which included most of the denitrifying bacteria. During the experiment, the nitrogen and phosphorus removal efficiency of AC-BRC and ACI-BRC were enhanced obviously, but ammonia nitrogen accumulated in ACI-BRC in the early stage of the reaction.