The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy (CLSM) and also provides selective excitation of a smaller focal volume with greater depth localization. E. coli PHL628 biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Active biomass fractions were observed at all depths in biofilms (up to 350 µm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness, and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamido-quinoline) was synthesized, and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 µm) biofilm, but was located only at the surface of thick biofilms, penetrating less than 20 µm after 1 hour exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm.