Metagenomics and bacterial culture were used to determine the normal skin microbiome of the Western mosquitofish (Gambusia affinis). This is the first study of G. affinis, and the most in-depth study of any fish skin, utilizing a combination of 16S profile pyrosequencing and culture analysis. Over 1800 sequences obtained from three individuals reveal that over half of all sequences come from five invariant genera, Acinetobacter, Sphingomonas, Acidovorax, Enhydrobacter, and Aquabacterium. The microbiome is diverse but has low equitability, with a total of 81 genera detected. Challenge studies suggest that non-native bacteria cannot colonize the skin. This definition of the normal skin microbiome lays the foundation for future studies with this model system.