Although nitrogen (N) enrichment enhances both soil N availability and soil acidification, it is difficult to isolate their effects on microbial taxa that drive the soil carbon (C) dynamics under different microbial C limitation conditions in natural ecosystems. Based on long-term field N addition, field acid addition, and laboratory incubation experiments in the Inner Mongolian grassland, we disentangled the effects of increased N availability and soil acidification on relative abundance of bacterial and fungal taxa under the mild (soil incubation for 28 days) and strong microbial C limitations (soil incubation for 191 days). Bacterial and fungal taxa are grouped into four ecological categories (N sensitive, acid sensitive, N and acid sensitive, N and acid insensitive) with multiple levels of N addition. In the context of the mild microbial C limitation, increased N availability promoted the relative abundance of the fungal class Eurotiomycetes (N positive sensitivity) under the low–medium levels of N addition, with an associated decrease in soil labile carbonyl C content. Conversely, increased N availability reduced the relative abundance of fungal class Sordariomycetes (N negative sensitivity) and bacterial gene copies (N and acid negative sensitivity) under the high levels of N addition, with associated increases in labile O-alkyl C and di-O-alkyl C content, making soil C more labile. In the context of the strong microbial C limitation, increased soil acidification promoted the abundance of acidophilous fungal classes Sordariomycetes and Eurotiomycetes under the low–medium levels of N addition, with an associated decrease in soil labile carbonyl C content. However, increased N availability promoted the relative abundance of the bacterial phylum Thaumarchaeota (N positive sensitivity equal to acid negative sensitivity), with associated low labile O-alkyl C and di-O-alkyl C content, leaving C chemistry more resistant. By applying the ecological category concept to soil microbes, our findings highlight that the N enrichment-induced shifts in abundance of N- and/or acid-sensitive categories are tightly associated with the changes in soil organic carbon (SOC) chemical composition, and the relationship between microbial function groups and SOC chemistry varied substantially under the mild versus strong microbial C limitations.