Colicins are narrow-spectrum antibiotics produced by and active against Escherichia coli and its close relatives. Colicin-producing strains cannot coexist with sensitive or resistant strains in a well-mixed culture, yet all three phenotypes are recovered in natural populations1. Recent in vitro results conclude that strain diversity can be promoted by colicin production in a spatially structured, non-transitive interaction2, as in the classic non-transitive model rock–paper–scissors (RPS). In the colicin version of the RPS model, strains that produce colicins (C) kill sensitive (S) strains, which outcompete resistant (R) strains, which outcompete C strains. Pairwise in vitro competitions between these three strains are resolved in a predictable order (C beats S, S beats R, and R beats C), but the complete system of three strains presents the opportunity for dynamic equilibrium2. Here we provide conclusive evidence of an in vivo antagonistic role for colicins and show that colicins (and potentially other bacteriocins) may promote, rather than eliminate, microbial diversity in the environment.