Eutrophication and lake depth are of key importance in structuring lake ecosystems. To elucidate the effect of contrasting nutrient concentrations and water levels on the microbial community in fully mixed shallow lakes, we manipulated water depth and nutrients in a lake mesocosm experiment in north temperate Estonia and followed the microbial community dynamics over a 6-month period. The experiment was carried out in Lake Võrtsjärv—a large, shallow eutrophic lake. We used two nutrient levels crossed with two water depths, each represented by four replicates. We found treatment effects on the microbial food web structure, with nutrients having a positive and water depth a negative effect on the biomasses of bacterial and heterotrophic nanoflagellates (HNF) (RM-ANOVA, p < 0.05). Nutrients affected positively and depth negatively the mean size of individual HNF and ciliate cells (RM-ANOVA; p < 0.05). The interactions of depth and nutrients affected positively the biomass of bacterivorous and bacteri-herbivorous ciliates and negatively the biomass of predaceous ciliates (RM-ANOVA; p < 0.05). Bacterivorous ciliates had lowest biomass in shallow and nutrient-rich mesocosms, whilst predaceous ciliates had highest biomass here, influencing trophic interactions in the microbial loop. Overall, increased nutrient concentrations and decreased water level resulted in an enhanced bacterial biomass and a decrease in their main grazers. These differences appeared to reflect distinctive regulation mechanisms inside the protozoan community and in the trophic interactions in the microbial loop community.