Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentousChloroflexibacteria but distal to the anammox bacterial cells. TheChloroflexibacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around theChloroflexi. The anammox S-layer protein was also abundant at junctions betweenChloroflexicells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentousChloroflexiinto a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a “public-good” EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox.