Pollution of the natural environment by microplastics has become a global issue in ecosystems as it poses a potential long‐term threat to biota. Microplastics can accrue in high abundances in sediments of aquatic ecosystems while also contaminating pelagic filter feeders, which could transfer pollutants up trophic webs. We assess the abundance and distribution of microplastics in benthic sediments and Cladocera taxa in a subtropical Austral reservoir using a combination of geospatial techniques, physicochemical analyses, diversity indices, and multivariate statistics between two seasons (i.e., hot–wet and cool–dry). We found particularly high densities of microplastics during the cool–dry season for both sediments (mean 224.1 vs. 189 particles kg–1 dry weight) and Cladocera taxa (0.3 particles per individual). Cladocera microplastic shapes were dominated by fibers with high densities of the transparent color scheme. Pearson correlation results indicated that sediment microplastic abundances were negatively correlated with chlorophyll–a concentration, temperature, and resistivity, whereas they were positively correlated with pH and salinity during the hot–wet season, with no variables significant in the cool–dry season. Cladocera microplastic abundances were positively correlated with conductivity and salinity during the cool–dry season, but no variables in the hot–wet season. These findings provide insights into the role of reservoirs as microplastic retention sites and the potential for uptake and transfer from lower trophic groups. These insights can be used to strengthen future monitoring and intervention strategies. Integr Environ Assess Manag 2024;1–15. © 2024 SETAC