Hyperhydricity is a physiological abnormality that frequently affects shoots that are vegetatively propagated in vitro. In this study, sugar beet (Beta vulgaris L. cv. Felicita) shoot tip explants were cultured on Murashige and Skoog medium supplemented with different concentrations of polyethylene glycol (PEG) 6000. We observed that higher concentrations of PEG 6000 and longer exposure (up to 4 wk) resulted in increasing levels of hyperhydration as well as browning and/or blackening of tissues in culture. A comparison of hyperhydric shoots with controls on the 28th day showed a marked increase in the content of water, phenolics, and malondialdehyde (MDA), which was positively correlated with an increase in the accumulation of PEG 6000. Selected antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (POX), and polyphenol oxidase (PPO) also increased in hyperhydric shoots, especially at lower concentrations of PEG 6000. Regression analysis indicated that strong linear relationships exist between SOD-APX (R (2) = 0.932), SOD-CAT (R (2) = 0.753), SOD-total phenolic content (R (2) = 0.966), APX-PPO (R (2) = 0.842), APX-total phenolic content (R (2) = 0.904), POX-CAT (R (2) = 0.751), and CAT-total phenolic content (R (2) = 0.806). Despite the correlation between different antioxidant enzymes and between the antioxidant enzymes and antioxidant compounds, was not able to prevent ROS damage in hyperhydric shoots. The negative correlation between SOD-MDA, POX-MDA, CAT-MDA, and MDA-total phenolics also indicated an increase in antioxidant enzyme activities, yet the increase in these antioxidant compound contents did not prevent lipid peroxidation of in vitro propagated beet shoots.