The current study was undertaken to elucidate the impact of the herbicide pretilachlor (3 µg ml-1 and 6 µg ml-1) on cyanobacteria, Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 under three levels of photoacclimatization (suboptimum, 25 µmol photon m-2 s-1; optimum, 75 µmol photon m-2 s-1; and supra-optimum, 225 µmol photon m-2 s-1) by analyzing certain physiological (biomass accumulation, photosynthesis, Chl a fluorescence and respiration) and biochemical parameters (photosynthetic pigments‒ chlorophyll a, carotenoids and phycocyanin; reactive oxygen species‒ O2•¯, H2O2, lipid peroxidation; antioxidant system‒ superoxide dismutase, peroxidise, catalase and glutathione-S-transferase). The light conditioning played the most prominent role in deciding the extent of herbicide toxicity on both the tested cyanobacteria as the maximum toxicity was observed in suboptimum light acclimatized cyanobacterial cells corroborated by the least growth in the same cells. The impact of pretilachlor treatment on photosystem II photochemistry viz. φP0, Ѱ0, φE0, PIABS, ABS/RC, TR0/RC, ET0/RC and DI0/RC was also altered by light acclimatization. The percent rise in oxidative stress markers (SOR and H2O2) and consequent lipid peroxidation (MDA equivalents) were also highest in suboptimum light acclimatized cells exposed to pretilachlor which could not be prospered with compatible antioxidant performance. Conversely, supra-optimum light acclimatized cells of both the cyanobacteria was found to accelerate the activities of all the studied enzymes and thus able to counterbalance the pretilachlor toxicity and supported the healthier growth.