Lake mixing disrupts chemical and physical gradients that structure bacterial communities. A transplant experiment was designed to investigate the influence of post-mixing environmental conditions and biotic interactions on bacterial community composition. The experimental design was 3 × 2 factorial, where water was incubated from three different sources (epilimnion, hypolimnion, and mixed epilimnion and hypolimnion) at two different locations in the water column (epilimnion or hypolimnion). Three replicate mesocosms of each treatment were removed every day for 5 days for bacterial community profiling, assessed by automated ribosomal intergenic spacer analysis. There were significant treatment effects observed, and temperature was the strongest measured driver of community change (r = −0.66). Epilimnion-incubated communities changed more than hypolimnion-incubated. Across all treatments, we classified generalist, layer-preferential and layer-specialist populations based on occurrence patterns. Most classified populations were generalists that occurred in both strata, suggesting that communities were robust to mixing. In a network analysis of the mixed-inocula treatments, there was correlative evidence of inter-population biotic interactions, where many of these interactions involved generalists. These results reveal differential responses of bacterial populations to lake mixing and highlight the role of generalist taxa in structuring an emergent community-level response.