A high-resolution survey of distribution, abundance and composition of phytoplankton was carried out for the first time in surface waters of the continental shelf off Chilean Patagonia (41–48°S). An Imaging FlowCytobot was used along the survey track to record phytoplankton in the size range of 10–120 μm during the austral spring of 2018. Phytoplankton community structure was complemented with continuous underway measurements of temperature and salinity, and physicochemical parameters of the water column at 35 oceanographic stations. Our results evidenced two main macrozones with distinctive phytoplankton assemblages delimited latitudinally at ~45°S. The northern macrozone was characterized by higher surface temperature and salinity, Si:N ratio > 1, diatoms of the genera Thalassiosira and Chaetoceros, and dinoflagellates accounting for over 70% of the total abundance. The southern macrozone, with lower surface temperature and salinity and Si:N ratio < 1, was characterized by members of the genera Guinardia, Lauderia and Cerataulina, representing over 60% of the total phytoplankton. These changes were attributable to the strong influence of freshwater at latitudes higher than 45°S and the enhanced discharge of meltwaters from Patagonian icefields in the area of the Taitao Peninsula and the Gulf of Penas (47–48°S). Fresh and cold waters impacted the water column stratification and the availability of dissolved silicic acid with potential effects on phytoplankton composition and diatom cell silicification and, thus, on carbon exportation. Our estimations of phytoplankton carbon were comparable to those observed in Patagonian fjords and the highly productive upwelling ecosystem of central Chile. We suggest that the continental shelf off Patagonia can contribute significantly to strengthen the biological carbon pump through the synthesis, exportation, and sequestration of phytoplankton-based organic carbon in the southeastern Pacific Ocean.