In agricultural watersheds, riparian buffer systems (RBS) planted with perennial vegetation such as trees, grasses, and shrubs host diverse soil microbial communities that drive ecosystem processes such as carbon and nitrogen cycling. Despite the huge role soil microbes play in controlling biogeochemical cycling, studies that characterized microbial communities in RBS are limited. Soil was sampled for microbial analysis on July 13, 2017, and biweekly between March and November 2018 from four RBS, undisturbed natural forest, coniferous forest, rehabilitated agroforest, grass buffer, and agricultural land. Greenhouse gases (GHG) such as nitrous oxide (N2O) and carbon dioxide (CO2) were measured using the static chamber technique. Root functional traits such as specific root length and root tissue density were determined. In a series of in-situ studies using cutting-edge molecular tools such as quantitative real-time PCR and high-throughput sequencing coupled with bioinformatic tools, microbial community structure was determined in relation to soil properties, GHG emissions, and root functional traits. Results indicate that soil microbial communities and N-cycling functional genes differed between land uses. In 2017, soil nitrate and moisture were the main drivers of N-cycling functional genes and N2O emissions. Non-metric multidimensional scaling analysis indicated significant differences in bacterial and fungal community composition between sites. In 2018, N-cycling genes were actively transcribed, the activity of nitrifier communities was found to be most responsive to changes in land use. Co-occurrence network analysis determined microbial hub taxa such as Xanthomonadaceae and Burkholderiaceae influential in maintaining the structure of microbial communities and identified microbial taxa that consistently predict N2O and CO2 emissions across the land-uses. Finally, a root exclusion and trait-based study found that bacteria, not fungi, diversity was impacted by root removal. Increased fungal nodes and bacteria-fungi interactions indicated a shift to a more fungi-dominated system when roots were removed. Distinct co-occurrence patterns were observed in grass buffer compared to the tree-dominated sites. Microbial taxa such as Gemmataceae was associated with root trait expressions; root traits are predictors of microbial taxa. These findings show that disentangling the mechanism of plant-soil–microbe interactions is crucial for developing science-based effective management strategies to mitigate GHG emissions in RBS.