The rearing of calves is probably the most critical and decisive phase of dairy farming, most of the problems occur due to errors in sanitary management, mainly during the creation, when the animals are considered more susceptible. It is during this period that different types of diseases arise, especially diarrhea, which is considered common in dairy herds in Brazil. The objective of the research was to evaluate the effect of ozone gas in the in vitro control of Enterobacterial strains isolated in Nelore calves with diarrhea. The study was carried out after submission and approval by the Ethics Committee for Animal Use (CEUA). Feces samples were collected from 14 calves with diarrhea, performed by digital rectal examination, using disposable gloves for each animal. The material was placed in a sterile universal collector. The feces were cultured and the culture media were inoculated, incubated and evaluated macro and micromorphological and conventional biochemical. The presence of enterobacteria was verified, being 11 samples of Escherichia coli and 3 of Salmonella sp. Bacterial suspensions were exposed to ozone gas at different times and concentrations. Subsequently, aliquots were seeded in triplicate on Mac Conckey Agar plates to determine the number of colony forming units (CFU). The results showed that the causative agents of diarrhea in calves were microorganisms Escherichia coli and Salmonella spp. Ozone gas promoted effective antimicrobial action in vitro of Salmonella sp. with 25 min and E. coli with 30 min of exposure to ozone gas.