Microbial biofilms are present in soils, sediments, and natural waters. They contain bioorganic metal-complexing functional groups and are thought to play an important role in metal cycling in natural and contaminated environments. In this study, the metal-complexing functional groups present within a suspension of bacterial cell aggregates embedded in extracellular polymeric substances (EPS) were identified in Zn adsorption experiments conducted at pH 6.9 with the freshwater and soil bacterium Pseudomonas putida. The adsorption data were fit with the van Bemmelen-Freundlich model. The molecular speciation of Zn within the biofilm was examined with Zn K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. The Zn EXAFS data were analyzed by shell-by-shell fitting and linear least-squares fitting with reference spectra. Zinc sorption to the biofilm was attributed to predominantly Zn-phosphoryl (85 ± 10 mol %) complexes, with a smaller contribution to sorption from carboxyl-type complexes (23 ± 10 mol %). The results of this study spectroscopically confirm the importance of phosphoryl functional groups in Zn sorption by a bacterial biofilm at neutral pH.