Plants from the genus Pterocaulon are popularly used as antifungal and wound-healing agents. Such activities have been related to coumarins, which are abundant in those plants. Coumarins are soluble in organic solvents, such as hexane and dichloromethane, and some of them are also soluble in hot water. Considering that infusion and decoctions of these plants are used in traditional medicine, the aim of this study was to identify and quantify the coumarins in the aqueous extract of Pterocaulon balansae. The aqueous extract was obtained by dynamic maceration and the compounds were characterized by UPLC-UV-MS analysis. A new coumarin and 5-methoxy-6,7-methylenedioxycoumarin, used for validation of the analytical HPLC method were obtained by partition of the aqueous extract with n-hexane. The HPLC method validated was linear, specific, and precise. Seven coumarins were characterized in the aqueous extract in a range of 0.584–54 mg/g of dry plant material. The main compound, 5,6-dimethoxy-7-(3′-methyl-2′,3′-dihydroxybutyloxy)coumarin, is described for the first time in P. balansae together with a new compound, 5,6-dimethoxy-7-(2′,3′-epoxy-3′-methylbutyloxy)coumarin.