This study evaluated the effects of calcium butyrate (CB) and bacitracin methylene disalicylate 50 (BMD) combined with different phytase concentrations on broiler intestinal health and physiology. Day-old YPM x Ross 708 male broilers (2,880) were distributed in 72 floor pens and assigned to 1 of 9 treatments (8 replicates/treatment). This experiment was a factorial arrangement including 2 phytase concentrations (500 or 1,500 FTU/kg) and 4 microbiota modulating feed additive levels (MMFA
(1) none, (2) only CB (0.5 g/kg of diet), (3) only BMD (55 mg/kg of diet), or (4) both CB and BMD). Additionally, a negative control without phytase and MMFA was included. Intestinal permeability was assessed on d 27. Jejunum wall and cecal content samples were collected on d 28 and 42 to assess jejunum villus height (VH), crypt depth, tight-junction and mucin gene expression, cecal microbiome diversity, and predicted bacterial metabolic pathways. Phytase and MMFA did not influence intestinal permeability (P >
0.05). Combining both CB and BMD with 1,500 FTU/kg of phytase compared to 500 FTU/kg lowered d 28 VH (P ≤ 0.05). Jejunal expression of CL-1, CL-4, CL-5, and ZO-2 on d 28 as well as CL-2 on d 42 changed between MMFA when combined with 1,500 FTU/kg of phytase but not 500 FTU/kg (P ≤ 0.05). Day 42 Pielou's evenness increased when 1,500 FTU/kg of phytase was combined with both CB and BMD compared to no MMFA (P ≤ 0.05). The cecal microbial beta diversity was not influenced by phytase, MMFA, or their interaction (P >
0.05). Overall, broiler intestinal health and physiology were influenced by CB and BMD depending on phytase concentration, demonstrating the complex interactions between these feed additives.