The N:P stoichiometry of a water body is one of the most commonly used indicators of its nutrient status and algal growth. However, in a dynamic aquatic ecosystem the N:P stoichiometry of phytoplankton is highly variable and depends on environmental conditions and key microbial interactions that influence their growth, such as grazing pressures and the microbial loop. Here we determine the influence of the nutrient-dependent microbial interactions between zooplankton, phytoplankton and bacteria on the ecological stoichiometry at different trophic levels and how they relate to water column properties. A 1D hydrodynamic–ecological model (DYRESM–CAEDYM) was applied to Lake Kinneret (Israel) for examining how the internal nutrient ratios of several phytoplankton functional groups correlate with nutrient ratios within the water column, and further explore how the microbial loop shapes the patterns of stoichiometry within the food web by testing two microbial loop configurations. The results showed that the average internal N:P ratios of the phytoplankton community followed their total carbon biomass patterns, and that seasonal patterns of simulated dissolved inorganic N to total P (DIN:TP) ratios in the water column were a useful indicator for reflecting the bulk phytoplankton N:P stoichiometry as compared with total N to total P (TN:TP) ratios and dissolved inorganic N to dissolved inorganic P (DIN:DIP) ratios. However, the internal N:P ratio patterns of individual phytoplankton groups did not necessarily correlate with DIN:TP ratio patterns in the water column. This was because different microbial processes regulate nutrient flows to individual phytoplankton groups. Our simulations with the microbial loop highlight the ability of bacteria to regulate phytoplankton stoichiometry. These results provide an improved mechanistic understanding of the food web in aquatic ecosystems.