Lost City is a unique off-axis hydrothermal vent field characterized by highly alkaline and relatively low-temperature fluids that harbours huge carbonate chimneys. We have carried out a molecular survey based on 18S rDNA sequences of the eukaryotic communities associated with fluid–seawater interfaces and with carbonates from venting areas and the chimney wall. Our study reveals a variety of lineages belonging to eight major taxa: Metazoa, Fungi, Heterokonta (Stramenopiles), Alveolata, Radiolaria, Cercozoa, Heterolobosea and Euglenozoa. We detected one fungal lineage that appears to be widespread in hydrothermal systems both submarine and continental. Alveolates were the most abundant and diverse group in Lost City samples, although their distribution was very different in carbonate, where ciliates dominated, and in fluid–seawater libraries, where dinoflagellates, Group I and Group II (Syndiniales) marine alveolates were profuse. Similarly, Euglenozoa also displayed a differential distribution, kinetoplastids being present on carbonates and a novel group of diplonemids so far exclusively observed in the deep sea being dominant in fluid–seawater libraries. Protist lineages identified in this ecosystem likely correspond to grazers, decomposers and parasites, playing key roles in the food web of the Lost City ecosystem.