Antibiotics are widely used to treat infections in humans. However, the impact of antibiotic use on host cells is understudied. Here we identify an antiviral effect of commonly used aminoglycoside antibiotics. We show that topical mucosal application of aminoglycosides prophylactically increased host resistance to a broad range of viral infections including herpes simplex viruses, influenza A virus and Zika virus. Aminoglycoside treatment also reduced viral replication in primary human cells. This antiviral activity was independent of the microbiota, because aminoglycoside treatment protected germ-free mice. Microarray analysis uncovered a marked upregulation of transcripts for interferon-stimulated genes (ISGs) following aminoglycoside application. ISG induction was mediated by Toll-like receptor 3, and required Toll/interleukin-1-receptor-domain-containing adapter-inducing interferon-β signalling adaptor, and Interferon regulatory factors 3 and 7, transcription factors that promote ISG expression. XCR1+ dendritic cells, which uniquely express Toll-like receptor 3, were recruited to the vaginal mucosa upon aminoglycoside treatment and were required for ISG induction. These results highlight an unexpected ability of aminoglycoside antibiotics to confer broad antiviral resistance in vivo.