Social insects use odors as cues for a variety of behavioral responses, including nestmate recognition. Past research on nestmate recognition indicates cuticular hydrocarbons are important nestmate discriminators for social insects, but other factors are likely to contribute to colony-specific odors. Here we experimentally tested whether external microbes contribute to nestmate recognition in red harvester ants (Pogonomyrmex barbatus). We changed the external microbiome of ants through topical application of either antibiotics or microbial cultures. We then observed behavior of nestmates when treated ants were returned to the nest. Ants whose external microbiome was augmented with microbial cultures were much more likely to be rejected than controls, but ants treated with antibiotics were not. This result is consistent with the possibility that external microbes are used for nestmate recognition.