Development of carbon nanotubes toward commercial antibacterial applications warrants the understanding of their interaction mechanism with bacterial cells. The antibacterial activity and mechanism of acid-functionalized single-walled carbon nanotube (AFSWCNT) coated paper was assessed for gram-positive Staphylococcus aureus and gram-negative Escherichia coli models of bacteria. Better activity towards gram-positive bacteria was observed, whereas the presence of an outer membrane makes gram-negative bacteria more resistant to cell membrane damage caused by AFSWCNTs. Based on measured cytoplasmic efflux materials of bacteria, X-ray photoelectron spectroscopy, and scanning transmission electron microscopy combined with electron energy-loss spectroscopy imaging studies, we found that the better antibacterial activity of AFSWCNTs toward gram-positive bacteria is attributed to not only direct physical contact and piercing action, but also molecular-scale interaction with surface functional groups of bacteria. The novel antibacterial mechanism of AFSWCNTs might bring a promising strategy to design new antibacterial materials against drug-resistant bacteria species.