This research studies whether photoprotection mechanisms are able to counterbalance the short-term effect of two herbicides, acifluorfen methyl (AFM) and paraquat (PQ), that generate photo-oxidative stress in different subcellular locations. Duckweed plants grown under three light intensities (high-, medium-, and low light), and consequently expressing three levels of photoprotection, were exposed to both herbicides under the same light regime. Oxidative damage induced by AFM originated mainly from the cytosolic accumulation of protoporphyrin IX, leading to a process of plasma membrane disruption, a progressive and slow degradation of ascorbate and photosynthetic pigments, and glutathione accumulation. As most photoprotective mechanisms (antioxidants and xanthophylls-cycle-related energy dissipation) operate mainly within the chloroplast, these systems were unable to protect plants from AFM damage irrespective of the level of light acclimation. Paraquat effects developed more rapidly and to a greater extent than AFM in treated plants. Irrespective of the light intensity, the same sequence of degradation was observed: ascorbate followed by glutathione, α-tocopherol, pigments, and membrane disruption. In PQ-treated plants the generation of oxidative stress occurred mainly in the chloroplast, and cellular damage developed more slowly in highly photoprotected plants (high light); in fact, electrolyte leakage can be used as a marker for PQ tolerance. The effects of both herbicides indicate that the xanthophyll cycle is an early protective mechanism and confirms the central role of ascorbate in early photoprotection response. High levels of lipophilic and hydrophilic antioxidant contents did not lead to attenuated phytotoxicity of acifluorfen methyl and thus are not the basis to explain differential susceptibilities among duckweed plants.