We assessed the phototoxicity of a series of xanthene derivatives against E. coli, S. aureus, and S. cerevisiae, measured the physicochemical properties of the photosensitizers, and found the relationship between them. Without illumination, the dyes tested showed almost the same level of inherent toxicity to the same organism, which showed the inherent toxicity of dyes was primarily dependent on the structure of parent molecule. Upon illumination, the photosensitizers showed obvious phototoxicity to all organisms. The dyes showed stronger phototoxicity to Gram-positive bacteria. With the increasing number of halogen substituents, the singlet oxygen yields increased and the phototoxic activity increased too. There was no obvious correlation between relative lipophilicity and activity in the current study. Our results showed xanthenes had the potential to act as alternatives to conventional antimicrobial compounds and also could be used for the decontamination of microbially polluted waters.