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Urbanization can influence many environmental factors that can affect the condition, immunity, and gut microbiota of birds. Over the past several decades, the Galápagos Islands of Ecuador have experienced increasing human activity, which has led to recent changes in the morphology, gut microbiota, and immunity of Darwin’s finches. However, these traits have not been characterized before the exponential growth of human population size and tourist visitation rates, i.e., before 2009. The goal of this study was to determine the effect of land use on the fecal microbiota, immune response, and body measurements of Darwin’s finches in 2008, at a time of rapidly increasing human activity on the islands. Specifically, we compared fecal microbiota (bacterial diversity, community structure and membership, and relative abundance of bacterial taxa), proxies of immunity (lysozyme activity and haptoglobin, complement antibody, and natural antibody levels), and body measurements (body mass and condition, tarsus length) across undeveloped, agricultural, and urban areas for medium ground finches (Geospiza fortis) and small ground finches (G. fuliginosa). Lysozyme activity was lower and observed bacterial species richness was higher in urban areas compared to non-urban areas across both finch species. In medium ground finches, four genera (Methylobacterium-Methylorubrum, Escherichia-Shigella, Brucella, and Citrobacter spp.) were higher in urban areas compared to undeveloped areas. In small ground finches, Paucibacter, Achromobacter, Delftia, Stenotrophomonas, and Brucella spp. had higher relative abundances in undeveloped and agricultural areas whereas the genus Cutibacterium was more abundant in finches from urban and agricultural areas than in finches from undeveloped areas. Medium ground finches were smaller in undeveloped areas compared to the other two areas, but body mass of small ground finches did not differ across areas. Our results suggest that human activity can have an impact on immune measures and gut microbiota of Darwin’s finches.