The composition and distribution of the phytoplankton community on the southweastern Brazilian continental shelf and continental slope were evaluated through chemotaxonomic analyses (HPLC-CHEMTAX). The samples were collected during five oceanographic cruises from 2012 to 2014 in spring and autumn. Fourteen surface blooms of Trichodesmium (slicks) were registered during the field surveys. The Trichodesmium biomass was estimated by both pigment analyses and microscope counts, with significant correlation found between the methods, except in some slicks. However, myxoxanthophyll, a Trichodesmium pigment biomarker generally used in CHEMTAX, was detectable only in seven samples from stations with Trichodesmium slicks, indicating that their use in pigment chemotaxonomic approaches should be taken with caution, because it could lead to errors in the estimation of Trichodesmium biomass. Four water masses were identified at the surface: Plata Plume Water (PPW), Subtropical Shelf Water (STSW), Shelf Water (SW) and Tropical Water (TW). The identified water masses seem to be the main driving factor influencing the composition, distribution and biomass of phytoplankton groups in the study region. The salty-warm oligotrophic TW, associated with the Brazil Current, was the dominant surface water mass in both autumn and spring. The surface waters in this work were generally associated with low values of both total chlorophyll a (TChl a) and nutrient concentration. In no slicks conditions, surface TChl a concentration was low ranging between 0.04 and 2.44 mg m−3 (mean 0.27 mg m−3) in almost all oceanographic stations. The highest biomass concentrations were recorded in stations under influence of both PPW and STSW. Overall, Prochlorococcus, haptophytes, Synechococcus, and Trichodesmium dominated the phytoplankton community and their contributions were above 70% to total Chl a in both spring and autumn. The other phytoplankton groups (prasinophytes, cryptophytes and dinoflagellates) represented a minor fraction, with values below 15% of the total biomass. A Canonical Correspondence Analysis showed that Prochlorococcus, haptophytes, Synechococcus and Trichodesmium were related with both TW and SW and were also positively associated with weaker water column stability, a deeper mixed layer, temperature and salinity. In turn, cryptophytes, dinoflagellates, prasynophytes and diatoms, mainly associated with STSW and PPW, were positively correlated with higher stability and nutrients (nitrogen and phosphorus), and negatively associated with both temperature and salinity. Our results showed that the pigment analysis (HPLC-CHEMTAX) allowed a detailed mapping of phytoplankton communities' distribution in the south and southeastern Brazilian continental shelf and shelf-break, and that the Trichodesmium biomass, as currently estimated by this approach, should be carefully revised.