New detailed palynological and ostracodological analyses together with texture data from a sediment core drilled in Ostia Antica confirm the existence of the ancient Ostia harbour and its location by the Tiber River. Using the different proxies analysed in this work and chronologically framing the sediment record with three AMS radiocarbon dates, four phases have been singled out: pre-harbour, harbour bay under fluvial influence, more protected harbour basin and post-harbour phase. Ostracodology is used to reconstruct the marine versus freshwater influence in the basin. Palynology is used to reconstruct the plant landscape and the surrounding environment. Phases with low pollen concentration and expansions of NPPs suggest soil erosion and are alternated with quieter ones, where human impact was very clear. Deciduous oaks typical of coastal plain forests are the main taxon during the harbour phases. The occurrence of riparian trees increases in periods with low pollen concentration, high NPPs and very high pine percentages. These should be the periods in which important sediment inputs inside the harbour basin arrived and could be the expression of intense flooding phases. The comparison between the ostracod assemblages recovered in the two cores and has led to speculate a complex harbour structure. A separation could explain the micropalaeontological differences between the cores. Thus, we suggest that a pier must have been built in order to protect the inner harbour from the marine influence and to unload the goods transported by the big ships.