Host mucosal barriers possess an arsenal of defense molecules to maintain host-microbe homeostasis such as antimicrobial peptides and immunoglobulins. In addition to these well-established defense molecules, we recently reported small RNAs (sRNAs)-mediated interactions between human oral keratinocytes andFusobacterium nucleatum (Fn), an oral pathobiont with increasing implications in extra-oral diseases. Specifically, uponFninfection, oral keratinocytes releasedFn-targeting tRNA-derived sRNAs (tsRNAs), an emerging class of noncoding sRNAs with gene regulatory functions. To explore potential antimicrobial activities of tsRNAs, we chemically modify the nucleotides of theFn-targeting tsRNAs and demonstrate that the resultant tsRNA derivatives, termed MOD-tsRNAs, exhibit growth inhibitory effect against variousFntype strains and clinical tumor isolates without any delivery vehicle in the nanomolar concentration range. In contrast, the same MOD-tsRNAs do not inhibit other representative oral bacteria. Further mechanistic studies uncover the ribosome-targeting functions of MOD-tsRNAs in inhibitingFn. Taken together, our work provides an engineering approach to targeting pathobionts through co-opting host-derived extracellular tsRNAs.