To assess how microbial processing of organic C inputs to forest soils may be influenced by elevated CO2 and altered N dynamics, we followed the fate of 13C-labeled substrates in soils from the Duke Free Air Carbon Enrichment site where differences in soil N status have been imposed by 7 years of N amendments. Heterotrophic respiration and δ13C of respired CO2-C and phospholipid fatty acids (PLFA) were measured to track activities of microbial groups and estimate a relative measure of substrate use efficiency (PLFA-based SUE). Results indicate an increased proportion of fungal and actinomycete activity in elevated CO2 soils, which varied with substrate. The negative effect of N on vanillin phenolic-C incorporation into actinomycete PLFA suggests legacies of fertilization can mitigate increased C flow into actinomycetes with elevated CO2. Further, the fourfold increase in PLFA-based SUE for vanillin phenolic-C in elevated CO2 soils that received N suggests future enhanced N limitation in elevated CO2 soils may promote enhanced respiratory loss relative to incorporation of some C-substrates into microbial biomass. These short-term incubations did not reveal greater loss of soil organic carbon via respiration or shifts in SUE with elevated CO2. However, observed relative increases in activity of actinomycetes and fungi with elevated CO2 and mitigation of this effect on actinomycetes with N amendments suggests that elevated CO2 and predicted N limitation may alter the fate of slow-turnover soil organic matter (SOM) in two competing ways. Investigations need to focus on how these microorganisms may increase slow-turnover substrate use while possibly enhancing the prevalence of microbial cell wall structures that can serve as precursors of stabilized SOM.