The activity of soil microorganisms can be harnessed to promote agricultural sustainability. One such microbial approach, referred to as reverse fertilization, incorporates large quantities of high carbon:nitrogen (C:N) plant residues into the soil. The carbon amendment stimulates the growth of soil microorganisms and triggers microbial scavenging of soil nitrogen, resulting in nitrogen immobilization within microbial cells. Immobilized nitrogen is temporarily unavailable for plant uptake. Most research on this technique has involved controlling invasive plants to reestablish native plant communities that are sensitive to soils with high nitrogen fertility. The approach should be applicable to the management of agricultural weeds, particularly for nitrophilous species. We conducted one greenhouse and two field-based experiments exploring the potential of reverse fertilization as an agricultural weed management tool. We observed evidence of nitrogen immobilization in soils amended with high C:N amendments, including higher microbial biomass and respiration, and decreased plant-available soil nitrate. We also observed functional changes in the soil microbial community, primarily based on soil amendment treatment. Fungal and bacterial beta diversity were strongly influenced by the soil amendment treatment, with a consistent decrease in fungal alpha diversity in carbon amended soils. We also observed a consistent reduction in growth and competitive ability of nitrogen-responsive weeds in carbon amended soils compared to unamended soils. We found altered functional traits of the weed community based on soil amendment treatments in our field experiment. The results from the three experiments indicate the ability of nitrogen immobilization to become a useful component of an integrated weed management program, with increasing potential as digital agriculture integrates multiple and simultaneous ecological crop management options.