Manganese (Mn) toxicity can induce oxidative stress and impair photosynthesis in plants. The activity of antioxidant enzymes such as superoxide dismutase (SOD) is increased in Lolium perenne (perennial ryegrass) in response to Mn toxicity (mainly in tolerant cultivars), but it remains unclear whether non-enzymatic antioxidant compounds may have a role in Mn tolerance. Seedlings of perennial ryegrass cv. Nui (Mn-sensitive) and cv. Kingston (Mn-tolerant) were grown in a greenhouse in nutrient solution at increasing Mn doses over 21 days. Even though both cultivars showed similar Mn uptake, dry weight decreases and lipid peroxidation caused by excess Mn were higher in cv. Nui than in Mn-tolerant Kingston. Maximum quantum yield of photosystem II (PSII) (Fv/Fm) declined only in cv. Nui at the highest Mn dose. Effective quantum yield (Ф PSII), electron transport rate, CO₂ assimilation, and total chlorophyll concentration in leaves decreased under excess Mn, particularly in the sensitive cultivar. Interestingly, chlorophyll a/b ratio increased (indicating relatively lower concentration of light-harvesting chlorophyll proteins as an adaptive defence mechanism) with an increase in Mn supply only in cv. Kingston, which partially explained its greater Mn tolerance compared with Nui. Concentration of carotenoids was not directly associated with non-photochemical quenching values, suggesting that ryegrass did not dissipate an excess of absorbed energy under Mn toxicity by this mechanism. At increasing excess Mn, both enzymatic (SOD activity) and non-enzymatic antioxidant responses (radical scavenging ability and phenolic concentration) were enhanced, mainly in Kingston. The enhanced antioxidant response in this cultivar suggests the hypothesis of increased capacity to control Mn-triggered oxidative stress as reflected in the reduced lipid peroxidation.