The lower Miocene Estancia 25 de Mayo Formation consists of shallow-marine to estuarine deposits that bear numerous oyster shell beds distributed throughout the succession. Facies analysis reveals that oysters grew in the nearshore paleoenvironments of both the lower Quién Sabe and the upper Bandurrias members. Two oyster species were identified: Crassostrea(?) hatcheri, distributed in the lower two thirds of the column, and Crassostrea orbignyi, distributed in the upper third of the column within the transition from marine to the fluvial deposits of the overlying Santa Cruz Formation. Petrographic, cathodoluminiscence, and carbon and oxygen stable isotope analyses of individual growth increments were performed on carbonate from all the oyster beds, together with consecutive time-series analyses for one specimen of each species. The isotopic composition of the shell seems to be associated with the microstructure of the growth increments. Selective diagenetic alteration affected chalky growth increments; whereas translucent (foliated and prismatic) growth increments are well preserved and can be used to infer paleoenvironmental conditions. Isotopic data indicate that C.(?) hatcheri lived in normal marine waters with a range of paleotemperatures from 10.6 to 20.5 °C. Monospecific beds of this oyster are related to opportunistic and quick colonization of the sea bottom. More negative δ13C and δ18O values in C. orbignyi shells likely reflect decreased paleosalinities, and the resultant environmental stress controlled the development of these low-diversity oyster accumulations. The separate stratigraphic distribution of each species was therefore determined by the paleoenvironmental conditions.