Stable isotope probing (SIP) has been used to track nutrient flows in microbial communities, but existing protein-based SIP methods capable of quantifying the degree of label incorporation into peptides and proteins have been demonstrated only by targeting usually less than 100 proteins per sample. Our method automatically (i) identifies the sequence of and (ii) quantifies the degree of heavy atom enrichment for thousands of proteins from microbial community proteome samples. These features make our method suitable for comparing isotopic differences between closely related protein sequences, and for detecting labeling patterns in low-abundance proteins or proteins derived from rare community members. The proteomic SIP method was validated using proteome samples of known stable isotope incorporation levels at 0.4%, ∼50%, and ∼98%. The method was then used to monitor incorporation of (15)N into established and regrowing microbial biofilms. The results indicate organism-specific migration patterns from established communities into regrowing communities and provide insights into metabolism during biofilm formation. The proteomic SIP method can be extended to many systems to track fluxes of (13)C or (15)N in microbial communities.