The oligotrophy of the southern Adriatic Sea is characterized by seasonal stratification which enables nutrient supply to the euphotic layer. A set of interdisciplinary methods was used to elucidate the diversity and co-dependency of bacterio- and phytoplankton of the water column during the stratification period of July 2021. A total of 95 taxa were determined by microscopy: 58 diatoms, 27 dinoflagellates, 6 coccolithophores, and 4 other autotrophs, which included Chlorophyceae, Chrysophyceae, and Cryptophytes. Nanophytoplankton abundances were higher in comparison to microphytoplankton. The prokaryotic plankton community as revealed by HTS was dominated by Proteobacteria (41–73%), Bacteroidota (9.5–27%), and cyanobacteria (1–10%), while the eukaryotic plankton community was composed of parasitic Syndiniales (45–80%), Ochrophyta (2–18%), Ciliophora (2–21%), Chlorophytes (2–4%), Haptophytes (1–4%), Bacillariophyta (1–13%), Pelagophyta (0.5–12%) and Chrysophyta (0.5–3%). Flow cytometry analysis has recorded Prochlorococcus and photosynthetic picoeukaryotes as more abundant in deep chlorophyll maximum (DCM), and Synechococcus and heterotrophic bacteria as most abundant in surface and thermocline layers. Surface, thermocline, and DCM layers were distinct considering community diversity, temperature, and nutrient correlations, while extreme nutrient values at the beginning of the investigating period indicated a possible nutrient flux. Nutrient and temperature were recognized as the main environmental drivers of phytoplankton and bacterioplankton community abundance.