The vertical distribution of phytoplankton is of fundamental importance in the structure, dynamic, and biogeochemical pathways in marine ecosystems. Nevertheless, what are the main factors determining this distribution remains as an open question. Here, we evaluated the relative influence of environmental factors that might control the coexistence and vertical distribution of pico-nanoplankton associated with the OMZ off northern Chile. Our results showed that in the upper layer Synechococcus-like cells were numerically important at all sampling stations. Pico-nano eukaryotes and phototrophic nanoflagellates (PNF) also showed high abundances in the upper layer decreasing in abundance down to the upper oxycline, while only Prochlorococcus showed high abundances under oxycline and within the oxygen-depleted layer. Statistical analyses evidenced that temperature, oxygen, and carbonate chemistry parameters (pH and dissolved inorganic carbon, DIC) influenced significantly the vertical distribution of phototrophic pico-nanoplankton. Additionally, we experimentally-evaluated the combined effect of low pH/low O2 conditions on a nanophytoplankton species, the haptophyte Imantonia sp. Under control conditions (pH = 8.1; O2 = 287.5 μM, light = 169.6 μEm−2s−1), Imantonia sp. in vivo fluorescence increased over fifty times, inducing supersaturated O2 conditions (900 μM) and an increasing pH (8.5), whereas upon an experimental treatment mimicking OMZ conditions (pH = 7.5; O2 = 55.6 μM; light = 169.6 μEm−2s−1), in vivo fluorescence declined dramatically, suggesting that Imantonia sp. did not survive. Although preliminary, our study provides evidence about the role of low pH/low O2 conditions on the vertical distribution of nanophytoplankton, which deserve future attention through both fieldwork and more extended experimental experiences.