Flavothione (FT) and a series of 18 hydroxy- and methoxy-substituted flavothiones were screened for photobiological activity. The 5-hydroxy–substituted compounds (group 3) and the methoxy-substituted flavothiones were inactive. FT and the remaining hydroxy-substituted compounds, all displayed photobiological activity. Among these, the 3-hydroxy–substituted compounds (group 2) were the most efficient photosensitizers overall in spite of their concurrent fast photodegradation. FT and all other hydroxyflavothiones, not substituted in the 3- or 5-positions (group 1), were inefficient compared with group 2. Detailed photobiological tests were carried out for four flavothiones of groups 1 and 2. The biological tests included fungi, several strains of Escherichia coli, Salmonella typhimurium and mammalian cells. In addition, the ability of these flavothiones to perform lipid peroxidation was evaluated. FT and 6-hydroxyflavothione (group 1) induce DNA damage via H-atom abstraction from the lowest n, π* triplet state of the thione (oxygen independent). For 3-hydroxy and 3,6-dihydroxyflavothione (group 2), both DNA and the membrane are targets. The mechanism likely involves both energy transfer and electron transfer from the lowest π, π* triplet state to oxygen, to form singlet oxygen and the superoxide anion. Some of these compounds could be considered as models for environmentally safe photopesticides.