Constructed wetlands are multi-functional systems that can effectively store and transform pollutants primarily through natural processes. However, the removal of nitrogen pollutant by wetlands is highly variable, likely due to a combination of factors such as plant species-specific assimilation behavior, the effects of soil microbial diversity, and variable nitrogen inputs. In this study, the effects of plant species richness (i.e., number of plant species in a system) and seasonal nutrient loading (i.e., nitrogen fertilization) on the microbial community responsible for regulating nitrogen turnover in wetland mesocosm soils was investigated. Digital polymerase chain reaction was used to quantify bacterial abundance. Principal component analysis was employed to identify dominant patterns within the data, and resampling-based analysis of variance was used to assess statistical significance of any observed differences caused by fertilization, season, and/or plant species richness. Results indicated that fertilization or season, which was convolved with fertilization, was the dominant factor influencing the microbial community in the study environment. The effects of plant species richness were more nuanced. Its greater richness significantly impacted the abundance of only a subset of bacterial groups (i.e., the ammonia oxidizing bacteria, Nitrospira spp. of nitrite-oxidizing bacteria, and comammox, but not the denitrifying bacteria). Display omitted •Microbial community involved in nitrogen turnover was investigated.•Digital polymerase chain reaction was used to quantify bacterial abundance.•Principal component analysis was employed to identify dominant patterns.•Seasonal fertilization was the dominant factor influencing the microbial community.•Plant species richness level only impacted a subset of bacterial groups.