The Western Tropical North Atlantic is a highly dynamic marine system where the Amazon River Plume generates a patchwork of environmental conditions that favor different phytoplankton groups. To study phytoplanktonic community structure in such heterogeneous conditions, we used a set of six standard ship-based measurements taken from oceanographic surveys between 2010 and 2021 to characterize different habitat types. We then utilized a variety of multiparametric approaches to examine phytoplankton biodiversity in the different habitats to assess the biological relevance of our delineated habitats. Our approach generated a consistent set of habitat types across cruises carried out in multiple seasons and years. Our phytoplankton community analyses revealed strong distinctions among habitats at higher taxonomic levels, and weaker contrasts at the species level, a pattern consistent with niche partitioning among similar species. We identified significant and consistent differences in phytoplankton community composition among habitats. The few apparent mismatches we found between phytoplankton community composition and habitat may reflect recent hydrographic changes driven by mixing and/or upwelling, and thus may be a useful index to biologically-relevant temporal variation. Our habitat classification approach is straightforward and broadly applicable in identifying biologically distinct areas within heterogeneous and dynamic regions of the ocean.