Predicting respiration from roots and soil microbes is important in agricultural landscapes where net flux of carbon from the soil to the atmosphere is of large concern. Yet, in riparian agroecosystems that buffer aquatic environments from agricultural fields, little is known on the differential contribution of CO2 sources nor the systematic patterns in root and microbial communities that relate to these emissions. We deployed a field-based root exclusion experiment to measure heterotrophic and autotrophic-rhizospheric respiration across riparian buffer types in an agricultural landscape in southern Ontario, Canada. We paired bi-weekly measurements of in-field CO2 flux with analysis of soil properties and fine root functional traits. We quantified soil microbial community structure using qPCR to estimate bacterial and fungal abundance and characterized microbial diversity using high-throughput sequencing. Mean daytime total soil respiration rates in the growing season were 186.1 ± 26.7, 188.7 ± 23.0, 278.6 ± 30.0, and 503.4 ± 31.3 mg CO2-C m–2 h–1 in remnant coniferous and mixed forest, and rehabilitated forest and grass buffers, respectively. Contributions of autotrophic-rhizospheric respiration to total soil CO2 fluxes ranged widely between 14 and 63% across the buffers. Covariation in root traits aligned roots of higher specific root length and nitrogen content with higher specific root respiration rates, while microbial abundance in rhizosphere soil coorindated with roots that were thicker in diameter and higher in carbon to nitrogen ratio. Variation in autotrophic-rhizospheric respiration on a soil area basis was explained by soil temperature, fine root length density, and covariation in root traits. Heterotrophic respiration was strongly explained by soil moisture, temperature, and soil carbon, while multiple factor analysis revealed a positive correlation with soil microbial diversity. This is a first in-field study to quantify root and soil respiration in relation to trade-offs in root trait expression and to determine interactions between root traits and soil microbial community structure to predict soil respiration.