The contribution of carbohydrates to non-enzymatic processes such as glycation/autoxidation has been extensively investigated over the last decades. This may be attributed to either beneficial or detrimental effects of reducing carbohydrates, and most studies in the field of glycoxidation are focused on glucose. Non-enzymatic reactions of fructose have not been as thoroughly investigated as those of glucose. To compare glucose and fructose involvement in the generation of glycoxidation products under experimental conditions close to the physiological situation, we used intact Saccharomyces cerevisiae cells as in vivo model and cell-free extracts prepared from whole yeast cells as in vitro model. Both intact cells and cell-free extracts were incubated with glucose or fructose. It was shown that: (i) in vitro fructose was more reactive than glucose and produced higher level of autoxidation and glycation products; (ii) no substantive differences were observed for the effect of glucose and fructose on the intracellular level of glycoxidation products, when intact yeast cells were exposed to the high concentration of hexoses; (iii) the activity of defensive enzymes (superoxide dismutase, catalase, glyoxalases, and glutathione reductase) was increased in both glucose- and fructose-stressed yeasts, indicating the development of oxidative/carbonyl stress; (iv) glucose-6-phosphate dehydrogenase activity significantly dropped in yeast exposed to both hexoses, demonstrating its high sensitivity to reactive oxygen and carbonyl species; and (v) fructose more markedly activated glyoxalases than glucose. Involvement of glucose and fructose in the glycoxidation reactions as well as potential role of antioxidant and antiglycation enzymes in yeast protection against glycoxidation are discussed.