A species of marine diatom, Toxarium undulatum, has emerged as a problematic biofouler of contemporary environmentally benign marine coatings. Previous analyses by atomic force microscopy (AFM) showed the cell-substratum adhesive of this alga contained macromolecules with a modular protein backbone assembled into nanofibers in which the domains of the macromolecules folded and unfolded in a co-ordinated manner. In the present study, we investigated further the composition and properties of the adhesive. A combination of energy dispersive X-ray analysis (EDXA) and Fourier transform infrared (FTIR) spectroscopy showed that the adhesive contained mainly protein, carbohydrate, sulfate, calcium, and magnesium. AFM demonstrated that EDTA treatment of native T. undulatum adhesive resulted in rapid disruption of the adhesive nanofiber (ANF) structure but ANFs were restored by subsequent treatment (within 1 h) with solutions containing divalent cations. Prolonged exposure to EDTA (≥18 h) led to cell detachment. The soluble EDTA extract was separated from the cells, dialyzed, concentrated, and analyzed further. The extract had a protein-to-carbohydrate-to-sulfate weight ratio of 1.0 : 0.2 : 0.9 and contained a single, high-molecular-mass (>220 kDa) band by SDS-PAGE which was visualized by Stains-All® but not by Coomassie blue, indicating that it was a highly anionic macromolecule. The most abundant amino acids in the extract were glycine (22 mol%), aspartic acid/aspartamine (14 mol%), and histidine (11 mol%). The adhesive contained 11 neutral sugars dominated by mannose (50 mol%) and xylose (29 mol%). On the basis of these data, we propose that the ANFs of T. undulatum are composed of sulfated high-molecular-mass glycoproteins cross-linked by calcium and magnesium ions. The cross-linking enables domains of adjacent protein backbones to unfold and re-fold in register.