New photosensitizers are needed for photodynamic antimicrobial and anticancer chemotherapy. Two new groups of proflavine derivatives have been recently prepared and their action on the cancer cells has been investigated by our research team. In this paper, we studied an effect of UV-A irradiation of two groups of proflavines: 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs) and 1’,1”-(acridin-3,6-diyl)-3’,3”-dialkyldithiourea hydrochlorides (AcrDTUs) on a plasmid DNA (pDNA). These compounds induced a photocleavage of pDNA characteristic by generation of free radicals, single strand DNA breaks and formation of an open circular form of pDNA.