For almost three decades, flow cytometry has allowed researchers to investigate ocean planktonic communities using size and cell fluorescence properties. However, oceanographic applications must face two constraints. First, when dealing with marine microbes, instruments must be sensitive because these organisms are very small and with low fluorescence. Second, instruments must be portable to be used on board ships. We compared the performance of two instruments, the BD FACSCanto™ and BD Accuri™ C6. The former is an expensive laboratory‐based instrument which has a very good sensitivity, whilst the latter is less sensitive but presents critical advantages for field studies (easy handling and transportation, relatively low cost). We have analyzed 102 samples from the South Atlantic Ocean from three transects off Brazil, within the euphotic zone. We compared cell abundance of heterotrophic bacteria, Prochlorococcus and Synechococcus, as well as photosynthetic pico‐ and nano‐eukaryotes. Heterotrophic bacteria, pico‐ and nano‐eukaryotes could be easily detected with both cytometers. Prochlorococcus and Synechococcus populations were severely under‐estimated with the BD Accuri™ C6, particularly for samples from the well‐lit layers of the water column. Correction of abundance data using previously suggested approaches was not sufficient to fully compensate for the low sensibility. Our data suggest that the BD Accuri™ C6 is suitable for counting marine bacteria and photosynthetic eukaryotes, but not Prochlorococcus and Synechococcus.