The sediment-stabilizing effect of benthic diatoms was investigated in a laboratory setting. Axenic cultures of the benthic diatoms Nitzschia cf. brevissima and Cylindrotheca closterium were inoculated in Petri dishes containing sand and incubated under axenic conditions. By ensuring aseptic routines throughout the experiments, interference from other organisms occurring with diatoms in natural photothrophic biofilms was avoided. This allowed the examination of the role of benthic diatoms in sediment stabilization. Increases in the critical erosion shear stress of the sediment were observed in the presence of both diatom taxa relative to sterile sediment. However, N. cf. brevissima was more effective than C. closterium. Values of critical shear stress in the experimental system were in the same range as those observed in natural biofilms, which indicates that diatoms are important agents for biogenic stabilization. Extracellular carbohydrate contents in the microcosms were similar for both diatom species. However, in the presence of N cf. brevissima, extracellular carbohydrate correlated significantly to critical shear stress, explaining up to 80% of the variation, whereas this was not the case for C. closterium. Therefore, it was concluded that the quantity of extracellular polymeric substances (EPS) alone did not explain the biogenic stabilization. Observed adsorption of EPS to sediment particles depended on the relative amount of uronic acids in the exopolymers. Using fluorescently labeled lectins, confocal laser scanning microscopy showed that EPS secretion by N. cf. brevissima resulted in ordered three-dimensional matrix structures. It is suggested that the structuring of EPS plays an prominent role in the process of biostabilization, and that diatoms such as N. cf. brevissima are actively involved in producing the structure of EPS, whereas others such as C. closterium do not do so to the same extent.