Carbon nanotubes (CNTs) hold promise in manufacturing, environmental, and biomedical applications, as well as food and agricultural industries. Previous observations have shown that CNTs have antimicrobial activity; however, the impact of CNTs to human gut microbes has not been investigated. Here, the antibacterial activity of CNTs against the microbes commonly encountered in the human digestion system—L. acidophilus, B. adolescentis, E. coli, E. faecalis, and S. aureus—are evaluated. The bacteria studied include pathogenic and non-pathogenic, gram-positive and negative, and both sphere and rod strains. In this study, CNTs, including single-walled CNTs (SWCNTs, 1–3 μm), short and long multi-walled CNTs (s-MWCNTs: 0.5–2 μm; l-MWCNTs: >50 μm), and functionalized multi-walled CNTs (hydroxyl- and carboxyl-modification, 0.5–2 μm), all have broad-spectrum antibacterial effects. Notably, CNTs may selectively lyse the walls and membranes of human gut microbes, depending on not only the length and surface functional groups of CNTs, but also the shapes of the bacteria. The mechanism of antibacterial activity is associated with their diameter-dependent piercing and length-dependent wrapping on the lysis of microbial walls and membranes, inducing release of intracellular components DNA and RNA and allowing a loss of bacterial membrane potential, demonstrating complete destruction of bacteria. Thin and rigid SWCNT show more effective wall/membrane piercing on spherical bacteria than MWCNTs. Long MWCNT may wrap around gut bacteria, increasing the area making contact with the bacterial wall. This work suggests that CNTs may be broad-spectrum and efficient antibacterial agents in the gut, and selective application of CNTs could reduce the potential hazard to probiotic bacteria.