Microbial activity plays a critical role in determining the nutrient status of an ecosystem (i.e. N or C limitation). While the balance of C/N assimilation has been measured at the whole community scale, quantitative detection of N and C assimilation from a single substrate at the scale of individual taxa has not been carried out. We recently developed Chip-SIP, a microarray and NanoSIMS-based method for linking microbial phylogeny and function that allows simultaneous measurement of (15)N and (13)C incorporation. Here, we measured the relative incorporation of C and N from dual-labeled substrates by individual microbial taxa in bottle incubations of samples collected from an estuary. Incubation times < 24 h were sufficient to successfully detect active microbes incorporating (15)N ammonium. In subsequent experiments, we used the incorporation of labeled amino acids (AAs) as a proxy for heterotrophic activity and showed different levels of incorporation among different taxonomic groups. Taxon-specific differences in the net incorporation of AA-derived C and N indicate that the C/N relative use efficiency ranged from 0.8 to 1.4, where 1 reflects stoichiometric incorporation of C and N. Our results revealed that microbial organic matter processing is affected by taxon-specific physiological diversity, both in terms of general activity levels and in the ratio of assimilated C/N.