The use of antibiotics as growth promotors in the food animal industry has led to the generation of antibiotic resistant microorganisms, which are a major health risk to humans. Therefore, bans and limitations, including the FDA’s veterinary feed directive, have been placed on the use of antibiotics as growth promotors, and there has been a push to find an adequate alternative. In the laying hen, probiotics and/or prebiotics have shown promise as they help promote the colonization of beneficial bacteria in the GI tract of the host, increase feed efficiency, increase egg production, and provide protection against pathogenic bacteria. In this feeding trial, Leghorn HyLine W36 hens were fed diets supplemented with either 0.05% IMW50(prebiotic), 0.05% CALSPORIN (probiotic), 0.05% IMW50 and 0.05% CALSPORIN, or a control diet with no prebiotic or probiotic additive. The birds were maintained on this diet from 1 day to 66 weeks of age, over which time feed intake and egg production was measured. From 37 to 65 weeks of age, every 3-5 weeks, egg quality was evaluated through specific gravity, egg weight, albumen height, and eggshell breaking force measurements. At 6, 16, 32, and 64 weeks of age, the microbiota population in the ileal digesta was evaluated using T-RFLP analysis methods. Then at 64 weeks of age, splenic IFN-γ, IL-6, IL-10, and IL-12 expression was measured by RT-qPCR. Unexpectedly, fluctuations in feed disappearance were observed, potentially linked to factors such as changes in diet type, temperature variations, feed wastage, or a technical error. Birds on CALSPORIN-containing diets showed resilience and maintained egg production during a decline in feed disappearance, suggesting enhanced capabilities in digesting larger particle sizes. However, there were trade-offs, as egg quality parameters slightly decreased in supplemented diets. CALSPORIN alone showed a promising effect on survival probability. Microbial diversity in the ileum increased with CALSPORIN, but the IMW50 alone led dysbiosis, possibly impacting infection resistance. Additionally, the combined supplementation of IMW50 and CALSPORIN may have introduced a disruption in immunological homeostasis.