This article presents the results of oxygen stable isotope analysis on eastern oyster (Crassostrea virginica) samples (n = 25) from a Mission-Era (ca. AD 1565–1680) site, Pueblo Santa Catalina de Guale, on St. Catherines Island, GA. Stable isotope analysis was used to assess the seasonality of oyster harvesting on St. Catherines Island during the Mission Era to better understand how Spanish colonization and the Catholic mission system may have impacted Guale foodways. Five eastern oyster left valves were sampled from each known neighborhood of Pueblo Santa Catalina de Guale. Existing data regarding the seasonality of pre-colonial mollusk collection on St. Catherines Island and elsewhere in the coastal Southeast are discussed in conversation with the results of this analysis. This work indicates that Guale residents of St. Catherines Island during the Mission Era were harvesting oysters most intensely during cooler months. This pattern is consistent with estimates of pre-colonial shellfishing seasonality across the region and may even represent a modest increase in the seasonal constriction of shellfishing when compared to data from the preceding period. Salinity estimates based on the isotope data also suggest Guale people during the Mission Era were collecting from a comparatively narrower range of estuarine habitats. We argue that these subtle shifts are a consequence of the reorganization of Guale labor associated with missionization.