The evolutionary arms race between phytophagous insects and their hosts has long fascinated biologists. Plants and fungi evolved a wide variety of secondary chemicals that are hypothesized to provide defense. While these compounds render them distasteful or toxic to many herbivores, some insects retain the ability to use them as hosts. Understanding the mechanisms that allow phytophagous insects to consume chemically defended hosts remains an active area of research, and recent studies have begun to highlight the role of the gut microbiome in assisting with toxin metabolism. In this study, we examine the potential contribution of the gut microbiome to toxin tolerance in mushroom-feeding Drosophila in the immigrans-tripunctata radiation. These flies and their larvae are broadly polyphagous on a wide range of fleshy mushrooms including toxic Amanita species that produce cyclopeptide toxins. Very little is known about the mechanism(s) of toxin tolerance in the immigrans-tripunctata radiation beyond the fact that its species do not have mutations that would inhibit the toxins’ mode of action. To assess the role of the microbiome in detoxification, we reared larvae of six tolerant species on diets with and without the toxin α-amanitin after significantly altering their microbiome. To quantify the contribution of the microbiome, we measured several performance phenotypes, including survival to adulthood and thorax length. Our results demonstrated that the gut microbiome does not play a critical role in cyclopeptide tolerance within the immigrans-tripunctata radiation.