Port Curtis, a major shipping port, has undergone significant expansion in the last decade, with plans for further development into the future. These activities may result in an increase of contaminant concentrations, threatening local wildlife including sea turtles. This study used a species-specific in vitro bioassay to examine spatial and temporal differences in exposure to, and effects of, organic contaminants in green sea turtles foraging in Port Curtis. Blood was collected from 134 green sea turtles (Chelonia mydas) from five locations in the port over four years. Organic contaminants were extracted from blood, and the cytotoxicity of the extracts to primary green sea turtle cells was assessed. Results indicated spatially similar chemical contamination throughout Port Curtis, at levels significant to sea turtle health, and with signs that chemical contamination may be increasing over time. These results can provide valuable information on the health of green turtles as further development occurs.