The phyllosphere and soil are dynamic habitats for microbial communities. Non-pathogenic microbiota, including leaf and soil beneficial bacteria, plays a crucial role in plant growth and health, as well as in soil fertility and organic matter production. In sustainable agriculture, it is important to understand the composition of these bacterial communities, their changes in response to disturbances, and their resilience to agricultural practices. Widespread pesticide application may have had non-target impacts on these beneficial microorganisms. Neonicotinoids are a family of systemic insecticides being vastly used to control soil and foliar pests in recent decades. A few studies have demonstrated the long-term and non-target effects of neonicotinoids on agroecosystem microbiota, but the generality of these findings remains unclear. In this study, we used 16S rRNA gene amplicon sequencing to characterize the effects of neonicotinoid seed treatment on soil and phyllosphere bacterial community diversity, composition and temporal dynamics in a 3-year soybean/corn rotation in Quebec, Canada. We found that habitat, host species and time are stronger drivers of variation in bacterial composition than neonicotinoid application. They, respectively, explained 37.3, 3.2, and 2.9% of the community variation. However, neonicotinoids did have an impact on bacterial community structure, especially on the taxonomic composition of soil communities (2.6%) and over time (2.4%). They also caused a decrease in soil alpha diversity in the middle of the growing season. While the neonicotinoid treatment favored some bacterial genera known as neonicotinoid biodegraders, there was a decline in the relative abundance of some potentially beneficial soil bacteria in response to the pesticide application. Some of these bacteria, such as the plant growth-promoting rhizobacteria and the bacteria involved in the nitrogen cycle, are vital for plant growth and improve soil fertility. Overall, our results indicate that neonicotinoids have non-target effects on phyllosphere and soil bacterial communities in a soybean-corn agroecosystem. Exploring the interactions among bacteria and other organisms, as well as the bacterial functional responses to the pesticide treatment, may enhance our understanding of these non-target effects and help us adapt agricultural practices to control these impacts.