We successfully prepared water-soluble fullerenol [C60(OH)46] that exhibited a high singlet oxygen quantum yield and efficiently generated reactive oxygen species. Additionally, the water-soluble C60(OH)46#nbsp;with a higher composition of exposed hydroxyl groups had superior two-photon stability and characteristics compared with that with a lower composition of such groups. Therefore, the prepared fullerenol can be an effective two-photon photosensitizer. The water-soluble C60(OH)46had favorable two-photon properties. During two-photon photodynamic therapy, the water-soluble C60(OH)46#nbsp;had substantial antimicrobial activity against Escherichia coli at an ultralow-energy level of 211.2 nJ pixel-1#nbsp;with 800 scans and a photoexcited wavelength of 760 nm.