The objective of this study was to evaluate the nutritional effects of single or combined feeding with Bacillus subtilis and a mixture of organic acids on egg production, intestinal ammonia concentration, ceacal microflora profile, and individual lipid fractions in chicken egg yolk. Two doses of B. subtilis were administered separately or together with an organic acid mixture composed of formic acid, fumaric acid, and sorbic acid. A total of 250 43-week-old Hy-Line Brown layers were divided into 5 groups with 5 replicates per group, and fed one of five experimental diets (control, maize-soybean meal-based diet without B. subtilis or organic acids; T1 – B. subtilis 1.0 × 10¹⁰ CFU/kg diet; T2 – B. subtilis 1.0 × 10¹⁰ CFU/kg diet + 0.15% organic acids; T3 – B. subtilis 2.0 × 10¹⁰ CFU/kg diet; and T4 – B. subtilis 2.0 × 10¹⁰ CFU/kg diet + 0.15% organic acids) for 8 weeks. Egg production and daily egg mass showed a raising trend or increased significantly in the group fed B. subtilis alone and the B. subtilis + organic acids combined groups, respectively. Additionally, the average eggshell strength in the treated groups was significantly higher compared to the control group. The abundance of ceacal lactic acid bacteria in the treated groups was significantly higher compared to the control group. The cholesterol content in egg yolk in all treated groups was significantly decreased compared to the control group. Overall, feeding B. subtilis exerted desirable effects on increasing egg production and eggshell quality, as well as modulating the ceacal microbial profile regardless of co-supplementation with organic acids.