Conspicuously colored dendrobatid frogs possess alkaloid-based antipredator defenses that are sequestered from a diet of arthropods. The type and quantity of alkaloids in dendrobatids vary substantially with geographic location, mainly due to differences in arthropod availability. It has been experimentally demonstrated that some individual alkaloids inhibit the growth of certain microbes, and that different alkaloids vary in their antimicrobial efficacy. We further tested this hypothesis by examining the antimicrobial effectiveness of naturally occurring mixtures of alkaloids (i.e., alkaloid cocktails) isolated from the dendrobatid frog Oophaga pumilio from five different locations in Costa Rica and Panama. Alkaloid cocktails in frogs from these locations varied significantly in their alkaloid composition. Bacterial cultures of Escherichia coli and Bacillus subtilis, and the fungus Candida albicans were subjected to alkaloid cocktails from individual frogs. These antimicrobial susceptibility tests demonstrated significant inhibition of bacterial and fungal growth of cultures incubated with these alkaloids, suggesting that the mixture of alkaloids present naturally in O. pumilio has the potential to defend frogs against natural microbes. Furthermore, there were significant differences in the degree of microbial inhibition among alkaloid cocktails, suggesting that frogs from different locations vary in their defense against microbes.