The Southern Ocean, a region characterized by high nutrient levels but often low productivity, hosts dynamic picophytoplankton communities crucial for its food web. This study investigated the spatial and inter-annual variability of picophytoplankton abundances and their environmental drivers in the Indian sector of the Southern Ocean during the austral summers of 2018 and 2020. Using flow cytometry for picophytoplankton quantification and standard oceanographic methods for environmental parameters (temperature, salinity, nitrate, phosphate, silicate), we employed descriptive statistics, inferential group comparisons (t-tests, analysis of variance), principal component analysis (PCA) and principal component regression (PCR) to analyse the dataset. Our analyses revealed significant differences in picophytoplankton abundances and environmental conditions across distinct oceanic fronts, between deep chlorophyll maximum and surface depths and, notably, between the two study years. PCA identified three major environmental gradients explaining over 93.5% of the variance in temperature, salinity, nitrate, phosphate and silicate. PCR confirmed our hypothesis: the abundance and carbon biomass of picoeukaryote II (PEUK-II) picophytoplankton was statistically significant overall (F-statistic = 3.415, P = 0.0290). The model explained 24.2% of the variance in PEUK-II abundance (R2 = 0.242), indicating its sensitivity to dynamic oceanographic conditions, with PC3 (primarily representing a salinity gradient) being a significant predictor. Conversely, Prochlorococcus-like/Synechococcus picophytoplankton abundance was not statistically significant overall (F-statistic = 2.068, P = 0.124), suggesting control by other, potentially non-linear factors. These findings highlight distinct ecological strategies among picophytoplankton groups and are vital for predicting their roles in the Southern Ocean’s microbial food web amidst ongoing environmental change.