Isotopic analysis of Micropogonias furnieri otoliths were used to get insight into palaeoceanographic conditions in the Guanabara Bay and Saquarema Lagoon, Rio de Janeiro state (RJ), located on the southeastern coast of Brazil, under upwelling influence of the Cabo Frio system. Archaeological otoliths come from two Holocene shellmounds (or sambaquis): Galeão and Beirada. For the first time, radiocarbon analysis using high accuracy techniques were performed at Galeão. Age range was determined to be between 5820 and 4980 cal BP, which extends the chronology of human settlement in the Guanabara Bay. Micro-samples of the otoliths were collected sequentially from the core to the edge, to provide continuous δ18O and δ13C isotopic profiles over the lifetime of the individual fish. Derived-δ18Ooto palaeotemperature estimates vary according to seasonality, resulting in a palaeoceanographic variation between 8 to 31°C for Guanabara Bay and 8 and 28°C for the Saquarema Lagoon. Our data indicate that whitemouth croakers were captured during the Middle Holocene from the Guanabara Bay and Saguarema Lagoon and resided in cooler temperatures compared to temperatures of current conditions.