Ozone is an allotropic form of oxygen, so in the medical field ozone therapy has special effects. Starting from the premise that bio-oxidative ozone therapy reduces the number of bacteria, in the present study two approaches were proposed: to evaluate the biological effects of ozone gas on the tooth enamel remineralization process and to demonstrate its impact on the morphology and confluence of human primary gingival cells, namely keratinocytes (PGK) and fibroblasts (HGF). The ozone produced by HealOzone was applied in vivo to 68 M1s (first permanent molars), both maxillary and mandibular, on the occlusal surfaces at pit and fissure. The molars included in the study recorded values between 13 and 24 according to the DIAGNOdent Pen 2190 scale, this being the main inclusion/exclusion criterion for the investigated molars. Because the gas can make contact with primary gingival cells during the ozonation process, both human gingival fibroblasts and keratinocytes were exposed to different doses of ozone (20 s, 40 s, 60 s), and its effects were observed with the Olympus IX73 inverted microscope. The contact of ozone with the human primary gingival cells demonstrates cell sensitivity to the action of ozone, this being higher in fibroblasts compared to keratinocytes, but it is not considered toxic because all the changes are reversible at 48 h after exposure.