Filamentous fungi and yeasts control cytoplasmic osmotic pressure through ion accumulation and synthesis of compatible osmolytes including polyhydric alcohols (polyols), proline, and trehalose. Authoritative data on the osmotic effects of these compounds were obtained using vapour pressure deficit osmometry. All osmolytes tested were characterised by nonlinear relationships between concentration and osmotic pressure. At high concentrations larger polyols generated higher osmotic pressures than smaller ones, though differences between the osmotic effects of polyols with three, four, five and six carbon atoms were not pronounced at lower (physiological) concentrations. Proline shared a similar relationship between concentration and osmotic pressure with polyols with five carbon atoms, while at concentrations above 0.5 M trehalose generated higher osmotic pressures than any of the polyols tested. Mixtures of trehalose and glycerol boosted osmotic pressure in a synergistic rather than additive fashion. These data provide new clues to the adaptive significance of glycerol accumulation, and also suggest that complex patterns of osmolyte synthesis are not due to differences between the osmotic effects of these compounds.