The gastrointestinal microbiome in animals provides an attractive target for manipulation to improve animal health and production performance. A better understanding of the chicken intestinal microbiome and how nutritional interventions can be used to modulate the microbiota is needed. Most studies of the intestinal microbiome of chickens have examined broilers with few studies focusing on the layer microbiome. This study focused on examining the impact of mannan-rich fraction (MRF) supplementation on the cecal microbiota of layers during and post peak lay. In a feeding trial, Shaver female laying hens were fed a control diet or a control diet supplemented with MRF in a randomized complete block design. Cecal content was collected from 10 randomly selected birds per treatment and subject to metagenome analysis at 4 timepoints (d 16, 32, 64, and 84 post-MRF introduction). Alpha diversity analysis revealed that Chao1 was significantly greater at D 16, D 32, and D 64 post-MRF supplementation but was significantly lower at D 84 in the MRF supplemented layers compared with the control (P < 0.005). PCoA plots showed that the bacterial community composition at the species level differed significantly (P < 0.001) between control and MRF supplemented layers at each timepoint. Microbiome analysis showed that following 84-days supplementation with MRF the pathogenic bacteria Listeria monocytogenes, Campylobacter jejuni, Enterococcus faecalis, and Clostridioides difficile were significantly lower in the layer cecum. In this study we observed greater alpha and beta diversity and lower bacterial pathogen detection over the 84-days following supplementation with MRF in laying hens. Increased bacterial diversity of the intestinal microbiota is one of the key determinants of colonization resistance against invading pathogens. With reference to the global challenge of antibiotic resistance and food security, reducing pathogenic bacterial species through the use of natural nonantibiotic alternatives is of particular importance for food chain integrity as well as flock health.