This study evaluated the effects of replacing zinc bacitracin with a symbiotic diet on the biochemical profile, liver development, and Clostridium perfringens presence in laying hens at 90 weeks of age. A total of 198 Dekalb–White hens were assigned to six experimental diets: MBM (corn, soybean meal, and meat and bone meal), BAC (MBM + 0.05% zinc bacitracin), and SIMC (MBM + 0.1% symbiotic), provided from day 1 to 90 weeks. Additional treatments, SIMR and SIMP, received symbiotics from weeks 6 and 17, re-spectively. At 90 weeks, blood and tissue samples were analyzed. Results showed that symbiotic-treated hens had 20% heavier livers (P < 0.01) and lower concentrations of uric acid, total proteins, AST, ALT, and LDH (P < 0.01) compared to BAC-treated birds. Phosphorus, triglycerides, and HDL levels were better in symbiotic groups. C. perfringens counts and antibiotic resistance (ampicillin, erythromycin, aminoglyco-sides, lincomycin, tetracycline, bacitracin) were significantly higher in BAC groups (P < 0.01). Symbiotic supplementation improved intestinal health, reduced pathogenic bacterial colonization, and enhanced liver function compared to continuous antibiotic use, especially when administered from early life stages.