Modern poultry production strives to ensure the safety of food products by constantly improving the health parameters of birds. Multi-strain probiotics containing effective microorganisms and microelements, especially zinc in chelated form, currently play an important role in poultry feeding. We hypothesized that supplementation of chicken embryos with a multi-strain probiotic and glycine-zinc chelate may influence lipid metabolism in the serum and tissues of newly hatched chicks, leading to an increase in the body's antioxidant capacity. Therefore, the aim of the study was to assess the lipid profile, fatty acid profile and malondialdehyde (MDA) concentration in the serum and tissues of newly hatched chickens supplemented in ovo with a multi-strain probiotic and zinc glycine chelate. The experiment was conducted on 1,500 fertilized hatching eggs obtained from 36-week-old commercial broilers (Ross x Ross 308). A multi-strain probiotic and zinc glycine chelate (Zn-Gly) were administered on the 17th day of incubation. Samples of peripheral blood and tissues of the liver, small intestine (ileum), pectoral muscle, thigh muscle and yolk sac, collected on the day of hatch and at 7 days post-hatch, were used to analyse biochemical parameters and determine the malondialdehyde level and fatty acid profile. The results indicate that in ovo supplementation with a multi-strain probiotic and Zn-Gly chelate on the 17th day of incubation affects fat metabolism. Simultaneous administration of a multi-strain probiotic and Zn-Gly chelate did not increase the concentration of polyunsaturated fatty acids (PUFAs) in the muscles. In birds treated with Zn-Gly chelate, the PUFA concentration was increased in the muscle tissue in the period up to 7 days post-hatch, i.e. during the development of the chicks. Moreover, the analysis of the results confirmed that in ovo supplementation with a multi-strain probiotic and Zn-Gly chelate stimulates the synthesis of saturated fatty acids (SFAs), mainly stearic, palmitic and myristic acids, and improves the ratio of PUFAs to SFAs. The multi-strain probiotic and Zn-Gly chelate administered in ovo did not cause excessive MDA synthesis in the tissues, suggesting that these preparations reduce oxidative stress in the developing embryo and newly hatched chick.