We investigated the controlling mechanisms of microbial and metazooplankton food‐web components at the surface‐ and bottom‐water layers of a thermally stratified floodplain lake (Lake Sakadaš, Kopački Rit floodplain, Croatia) during its connection with the river. Throughout the study, the lake was stratified continuously. The permutational multivariate analysis of variance provided evidence that only the surface‐water layer was impacted on by the flow and flood pulses, whereas the bottom‐water layer represented a zone of stable environmental conditions. The water depth was by one order of magnitude more important in determining environmental variables and plankton abundance than the hydrological phases. Under such circumstances, path analysis provided evidence that the planktonic food‐web interactions were strongly influenced by the sample layer, regardless of the flooding conditions. The transfer of organic matter to higher trophic levels at the surface was dominated by herbivory and, to a lesser extent, by predation, whereas bottom‐water layer was represented by additional routes (bacterivory and omnivory). The transfer of organic matter was different through the investigated water layers, of which the surface‐water layer was additionally structured by the floodplain hydrology.