[Background] Clostridium perfringens has been a major pathogen affecting the poultry industry, and its infection rate has been on the rise since the use of antibiotics was restricted. Bacteriophages with unique antibacterial effects and safety without residues have become a primary approach to reduce the use of antibiotics. [Objective] To evaluate the synergistic effects of Clostridium perfringens phage, Salmonella phage, and Bacillus subtilis on laying hens by analyzing the histological changes of the intestinal structure, the reduction of C. perfringens, and microbial diversity, so as to provide a scheme for the prevention and control of bacterial diseases in poultry with antibiotic reduction. [Methods] A total of 105 260-day-old Hyline Brown hens were selected and randomized into 5 groups of A: Clostridium perfringens phage SD72, B: SD72+Bacillus subtilis, C: Salmonella phage JDF1-6, D: SD72+JDF1-6+ Bacillus subtilis, and E: blank control. After the hens were treated for 42 days, the cecum contents were collected to determine the number of C. perfringens, and the intestinal structure was observed. The 16S rRNA gene amplicon was sequenced to reveal the intestinal microbial community structure, diversity, and metabolism. [Results] Compared with the blank control group, D group showed decreased number of C. perfringens (by 1.48 log₁₀ (CFU/mL)), decreased depth of jejunal crypts (P<0.05), and increased villus height/crypt depth (P<0.05). Firmicutes and Proteobacteria were dominant in cecum contents of laying hens in all the treatment groups after drinking water, and Lactobacillus(11.26%) had higher relative abundance at the genus level. The serum levels of IgA, IgG, and IgM were significantly increased (P<0.05), and intestinal microbial genes were mainly enriched in the genetic information processing pathways. The abundance of enriched genes in excretory system and immune disease-related pathways in group D was 755.95% higher than that in the blank control group. [Conclusion] The combination of mixed phages and B. subtilis could improve the intestinal health and immunity of laying hens by promoting the expression of microbial genes related to immune disease and improving the immunometabolism of intestinal flora. The findings provide a theoretical basis and biological resources for the prevention and control of bacterial diseases in poultry with reduced antibiotics.