The crystal of spiculisporic acid, [4S, 5S-(4,5-dicarboxy-4-pentadecanolide)], is orthorhombic, and the space group is P2₁ 2₁ 2₁ with Z = 4 at 23°C. The cell dimensions are as follows, a = 43.82 (1) Å, b = 7.540 (2) Å, c = 5.579 (2) Å, V = 1843 (1) ų. The molecules form intermolecular networks linked by hydrogen bonds around their polar carboxylic moieties. The hydrophobicity of the lactone ring (γ-butanolide) was evaluated at ca. 4 methylene linkages of n-alkyl chain from both of the molecular alignmental data and the surface active properties of sodium spiculisporates. Then, it was made clear that sodium spiculisporates, anionic polyfunctional biosurfactants, exerted a distinctive characteristic of lower critical micelle concentration (cmc) and γ(cmc) (surface tension at the cmc), simultaneously, through their properties. On the other hand, sodium salts of the lactone-cleaved derivative of spiculisporic acid (O-acid), showed large dispersing and calcium ion sequestration properties due to their plural polar heads.