Isomer-specific concentrations of nonylphenol (NP) and their predicted estrogenic potency were investigated in Sri Lankan waters for the first time. The total concentration of 13 NP isomers ranged from 90 to 1835 ng/L, while the predicted estrogenic equivalent concentration ranged from 0.072 to 1.38 ng 17β-estradiol (E2)/L. Bire Lake, located in the central area of the commercial capital, Colombo, had the highest contamination among the studied locations. These data show that NP levels in Sri Lankan waters are well within the recently reported concentrations in other regions of the world. The spatial differences in NP concentrations suggest that NP contamination in Sri Lanka may be widespread, and comprehensive study is vital.