An important feature of microbial communities is the spatial heterogeneity of extracellular chemistry. Scanning transmission X-ray microscopy (STXM) was used to map the spatial distribution of iron species throughout Pseudomonas aeruginosa biofilms to assess the influence of chemical heterogeneity on biomineralization. P. aeruginosa biofilms were treated with Fe(III)-amended media. Speciation and quantitative mapping using STXM image sequences in the Fe 2p3/2 (L3) absorption edge region revealed both Fe(II) and Fe(III) in localized microenvironments. Fe(III) was mainly associated with cell surfaces, while small amounts of Fe(II) was found in the extracellular space. Biofilms were also characterized using C 1s edge STXM image sequences. Anaerobic growth assays and confocal microscopy revealed the inability of P. aeruginosa to directly reduce Fe(III), implicating indirect iron reduction mechanisms in the formation of fine-grained, multivalent minerals. These studies suggest that geochemical microenvironments found throughout microbial communities are even more complex than previously believed.