Trace fossils documented in firm sediment and skeletal substrates are reported for the Middle Member of the Río Negro Formation (Upper Miocene-Lower Pliocene) in the northeastern Patagonia coastal zone of Argentina. Several trace fossils that demonstrate an intense organic activity of molluscs, mainly bivalves, annelids, and sponges, are described. In firm sediment, burrow structures are recorded on internal and composite molds of infaunal bivalves (Anadara, Scapharca, Dosinia, Chionopsis, Pitar, Dinocardium, Panopea) occurring in the basal unit of bioclastic sandstones of the early transgressive systems tract. The burrow structures are assigned to Arachnostega gastrochaenae and must have been produced either in the interface between valve and sediment fill or when the molds were exposed and consolidated on the bottom. Bioerosion structures on skeletal substrates (Aequipecten tehuelchus, Pododesmus camachoi) from the same unit are identified as Maeandropolydora sulcans and Centrichnus eccentricus. Bioerosion also occurs in oyster valves (Ostrea patagonica) preserved in mudstones of the late transgressive systems tract. Bioerosion includes borings of bivalves, sponges, and annelids and attachment scars of barnacles. The location of bioerosional structures, on both the external and internal surfaces of oysters, reveals that colonization by organisms occurred mainly post mortem. The bioerosion structures recorded suggest that the substrates were exposed to very low depositional rates. The trace-fossil assemblage is dominated by dwelling traces (Domichnia) followed by fixation traces (Fixichnia) and feeding traces (Fodinichnia).