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North American bison (Bison bison) are becoming increasingly important to both grassland management and commercial ranching. However, a lack of quantitative data on their diet constrains conservation efforts and the ability to predict bison effects on grasslands. In particular, we know little about the seasonality of the bison diet, the degree to which bison supplement their diet with eudicots, and how changes in diet influence gut microbial communities, all of which play important roles in ungulate performance. To address these knowledge gaps, we quantified seasonal patterns in bison diet and gut microbial community composition for a bison herd in Kansas using DNA sequencing-based analyses of both chloroplast and microbial DNA contained in fecal matter. Across the 11 sampling dates that spanned 166 days, we found that diet shifted continuously over the growing season, allowing bison to take advantage of the seasonal availability of high-protein plant species. Bison consumed more woody shrubs in spring and fall than in summer, when forb and grass intake predominated. In examining gut microbiota, the bacterial phylum Tenericutes shifted significantly in relative abundance over the growing season. This work suggests that North American bison can continuously adjust their diet with a high reliance on non-grasses throughout the year. In addition, we find evidence for seasonal patterns in gut community composition that are likely driven by the observed dietary changes.