Gaseous ozonation, an emerging technology, is applied to microbiological decontamination and to the degradation of residues and contaminants in wheat grains. However, due to its high oxidizing capacity, ozone may cause undesirable effects on the quality of grains or in their derivatives. This study aimed to evaluate the influence of ozone gas on wheat grain quality when exposed to different levels of ozone concentration, exposure time, and grain mass, using a full 2 3 factorial design. After the milling of ozonized grains, two fractions were evaluated: flour (A) and bran plus germ (B). In fraction A, the quality of the wheat flour was analyzed by both alveography and farinography. The flour extraction rate, falling number and gluten contents were also measured. The chemical and mineral profiles were determined in both fractions. A sensory evaluation, using the difference-from-control test, was applied to investigate the possible differences in aroma or in overall appearance of the flour obtained from the ozonized grains. The results showed that ozone concentration (60 mg/L) positively affected (p < 0.05) the toughness and the falling number of the flour. The other parameters of alveography, as well as farinography, gluten content, chemical composition, mineral, and sensory profiles were not affected (p > 0.05) by ozonation. This study demonstrated that gaseous ozonation, when applied under the conditions of 10 to 60 mg/L, from 2 to 5 h of exposure, grain mass from 2 to 5 kg, does not cause any negative impact on the wheat quality.