Interaction of pretilachlor with photosystem (PS)‐II of the cyanobacterium Desmonostoc muscorum PUPCCC 405.10 has been studied in this paper. Pretilachlor negatively affected growth, chlorophyll a (Chl a), photosynthesis, and carbon dissimilation in a dose‐dependent manner. Effects were also observed in PSs, especially PS‐II (an 11–35% decrease), as well as the whole photosynthetic electron transport activity. The fluorescence emission spectrum of Chl a revealed a dose‐dependent effect of pretilachlor on both the antenna and the core complex of PSs, with more severe effect on the former. Data of O‐J‐I‐P fluorescence transient of Chl a revealed that pretilachlor interfered with electron flow between QA and QB sites of PS‐II. It was further observed that pretilachlor decreased maximum fluorescence, variable and relative variable fluorescence, maximum quantum yield, quantum yield of electron transport, the rate of trapped exciton movement, quantum yield of electron transfer, and performance index of primary photochemistry; however, there was a progressive increase in the net rate of PS‐II closure, quantum yield of energy dissipation, and effective antenna size per active reaction center. A decrease in photosynthetic activity leads to a decrease in carbon dissimilation, as evidenced by low activity of glucose‐6‐phosphate dehydrogenase and pyruvate kinase. Thus, pretilachlor, which is otherwise known to kill weeds by interfering with cell division, affected the growth of the cyanobacteria by interacting with PS‐II.