The abuse of antibiotics and the release of nanoparticles (NPs) into the environment are of grave concern; however, their co-effect on the environment is not yet clear. In this study, the response of activated sludge exposed to ampicillin (AMP) and multi-walled carbon nanotubes (MWCNTs) was investigated in a sequencing batch reactor (SBR). The results showed that mixed liquor volatile suspended solid (MLVSS) values exposed to AMP and MWCNTs decreased to 83.48–90.53%, but the specific oxygen utilization rate (SOUR) was even higher with respect to the control for 24 h exposure. Compared to individual AMP, the co-occurrences of AMP and MWCNTs showed lower cytotoxicity to activated sludge, supported by a lower membrane permeability and higher cell metabolic viability. The microbial community structure changed when exposed to AMP and MWCNTs. Firmicutes phylum emerged in the top 20 phyla under the co-effect of AMP and MWCNTs, but not for AMP alone. It is worth noting that the relative abundances of antibiotic resistance genes (ARGs) under the co-effect of AMP and MWCNTs were significantly increased, especially bla_DHA. These results provide useful information for understanding the risk of the co-occurrences of antibiotics and CNTs, and can aid the sustainable and widespread application of novel carbon materials.