Biosurfactants have found widespread use across multiple industrial fields, including medicine, food, cosmetics, detergents, pulp, and paper, as well as the degradation of oil and fat. The culture broth of Aureobasidium pullulans A11231-1-58 isolated from flowers of Chrysanthemum boreale Makino exhibited potent surfactant activity. Surfactant activity-guided fractionation led to the isolation of three new biosurfactants, pullusurfactins A‒C (1‒3). Their chemical structures were established through the use of spectroscopic techniques, predominantly 1D and 2D NMR, in conjunction with mass measurements. We evaluated the surface tension activities of isolated compounds. At 1.0 mg l-1, these compounds showed high degrees of surfactant activity (31.15 dyne/cm, 33.75 dyne/cm, and 33.83 dyne/cm, respectively).