Nuclear magnetic resonance (NMR) is a noninvasive and nondestructive tool able to access several observable quantities in biofilms such as chemical composition, diffusion, and macroscale structure and transport. Pulsed gradient spin echo (PGSE) NMR techniques were used to measure spectrally resolved biomacromolecular diffusion in biofilm biomass, extending previous research on spectrally resolved diffusion in biofilms. The dominant free water signal was nulled using an inversion recovery modification of the traditional PGSE technique in which the signal from free water is minimized in order to view the spectra of components such as the rotationally mobile carbohydrates, DNA, and proteins. Diffusion data for the major constituents obtained from each of these spectral peaks demonstrate that the biomass of the biofilm contains both a fast and slow diffusion component. The dependence of diffusion on antimicrobial and environmental challenges suggests the polymer molecular dynamics measured by NMR are a sensitive indicator of biofilm function.