The spreading of antibiotic resistance in the modern world results from the application of uncontrolled and excessively intensive antibiotics for treating human infectious diseases and their use as growth stimulants in animal farming. In some economically developed countries, restrictions on the use of antibiotics are already applied in order to prevent the generation of resistant strains of microorganisms. An essential component of such means is antibiotic-free strategies in animal farming. Recently, strategies based on the alternative to both antimicrobials and growth promoters have been proposed, including probiotics, prebiotics, synbiotics, enzymes, and organic acids. Short-chain fatty acids and monoglycerides based on them are considered a promising alternatives to antimicrobial agents for poultry farming. However, the effects of these compounds on innate immunity and cytokine production in the intestinal system remain undiscovered. The experiment was carried out using two groups of Cobb 500 cross broilers in the conditions of an industrial poultry farm. The broiler chickens of the research group were exposed to a monoglyceride mixture with С3, С4, С8–С10 fatty acids from 1 to 7 days of life and 16 to 22 days of life at a dose of 0.5 l of the mixture per ton of water. On the 31-st day of life, productive parameters were evaluated, and five birds were selected from each group. The fragments of the duodenum, caecum, and rectum with contents were taken from the bird. In the presented study, the content of interferon-α in the tissue of the small intestine of broiler chickens under the conditions of drinking monoglyceride was studied. To evaluate the beneficial effect of monoglyceride, the state of the microbiome and productive indicators were determined. The study showed that the monoglyceride induces a statistically significant increase in the content of enterococcus in the duodenum, suppresses the growth of staphylococcus, and increases the production of interferon-α. Moreover, average daily weight gain relative to controls was almost 2% greater in monoglyceride-treated chicks. Thus, preservation of the state of microbiota and moderate stimulation of interferon-α production can be essential components of the multidirectional protective effect of monoglycerides and fatty acids.