Licensed under a Creative Commons Attribution 4.0 International LicenseAzodicarbonamide is an important chemical widely used in the industry as a blowing agent in the foam industry and in the food industry as a flour bleaching agent and a dough conditioner. The discussion about its biosafety is ongoing, in a number of countries, the use of azodicarbonamide is limited or even prohibited since a slightly carcinogenic effect of its by-products has been found. Despite this, many manufacturers continue to use it as a food additive. In a laboratory experiment, the effect of various doses of azodicarbonamide on the organism of laboratory animals under the background of a high-fat diet was determined by changes in their body weight, the state and mass indices of internal organs, blood parameters, the functional state of the nervous system, and changes in the intestinal microbiota. Four groups were formed from laboratory male rats, which for 21 days were consuming: a high-fat diet with the addition of 4%, 1%, 0.25%, 0% azodicarbonamide. It has been determined that azodicarbonamide did not cause a change in the organ mass index, but the addition of 4% and 1% of the substance to the diet significantly reduced the intensity of animals' body weight gain. Both excess fat in the diet and different doses of azodicarbonamide mainly caused functional disorders of the parenchymal organs, as evidenced by changes in the activity of blood enzymes (Aspartate aminotransferase, Alanine aminotransferase, De Ritis ratio (AST/ALT), Alkaline phosphatase) and protein metabolism (Total protein, Globulins, Protein coefficient, Urea). Significant changes in the physical and orientation activity and emotional status of the animals were not observed. Low concentrations of azodicarbonamide (0.25% and 1% of the feed mass) caused a pronounced decrease in the number of normal enzymatic properties of Escherichia coli strains below the control group and the reference range, and a high concentration (4%) significantly reduced the number of Lactobacillus bacteria in comparison with the control one but did not exceed references.