Ultraviolet B (UVB) radiation causes a number of harmful effects in cells including direct DNA damage, oxidative stress, signalling pathway disorders, inflammatory response and photocancerogenesis. Great effort is invested into the investigation of various natural compounds that can provide protection from the harmful effects of UVB radiation. Honey is a completely natural and nutritive product that contains flavonoids and phenols with antioxidant effect. Ultraviolet B radiation has been proven to be genotoxic, therefore the initial hypothesis of the study was that treating lymphocytes with honey can increase the survival rate and decrease primary DNA damage as well as the oxidative stress levels after exposure to irradiation. For this purpose, we used chestnut honey, which has been proven to have high ratios of polyphenols and good antioxidant activity. The study was conducted in vitro on the lymphocytes and peripheral blood plasma of a healthy male. There were two models of cells and whole blood exposure: (I) 2 hours of pretreatment with honey followed by exposure to UVB irradiation and an additional 1 hour incubation of cells/whole blood at 37 °C, (II) treatment including exposure to UVB irradiation and incubation of cells/whole blood with honey for 2 hours at 37 °C. The effects of the honey were analyzed at concentrations that correspond to the daily acceptable honey intake dose and a 10 times larger concentration than the daily acceptable dose. The primary damage of lymphocyte DNA, as well as the protective effect of honey, was evaluated using the alkaline comet assay, while the plasma samples were used to determine markers of oxidative stress. Chestnut honey showed good cytoprotective and genoprotective, as well as antioxidative effect after exposure of lymphocytes and whole blood to UVB irradiation. The beneficial effect of honey was more pronounced after the treatment of irradiated samples. The optimal concentration of honey was shown to be 1 mg/mL, while the lowest (0,2157 mg/mL) and the largest (10 mg/mL) concentrations showed a prooxidative effect in measuring oxidative stress markers.