This study was conducted to investigate the effects of dietary supplementation of a probiotic (Bacillus subtilis), acidifiers, and their combination on the mucosa of the broiler small intestine. A total of 220 straight run 1-day-old broiler chicks (Ross 308) were distributed into 4 experimental treatments with 5 replicates per treatment as a 2 × 2 factorial arrangement. The factors were probiotic and a blend of acidifiers with 2 levels: 0.0% and 0.05% for probiotic, and 0.0% and 0.2% for acidifiers. On day 42, tissue samples from the duodenum, jejunum, and ileum of 5 birds from each group were collected and processed for histology, immunohistochemistry, and scanning electron microscopy. Duodenal villus height (VH) increased (P ≤ 0.05) when probiotic was added to the diet. Chickens fed the probiotic had a significantly higher (P ≤ 0.05) percentage of intact villi compared to the other groups. The jejunum showed an increased VH in the group of birds supplemented with acidifiers. The ileum showed decreased (P ≤ 0.05) VH when probiotic was added to the diet, and increased VH when given acidifiers. Dietary supplementation of probiotic only or in combination with acidifiers showed a reduction (P ≤ 0.05) in the number of somatostatin immunoreactive cells (SIC) in the duodenal villi. A decrease in the number of SIC was also noted in the jejunal villi of birds receiving acidifiers. In conclusion, dietary supplementation with the probiotic, B. subtilis, or acidifiers improved the mucosal morphology of the small intestine in the broilers used in the present study. The 2 additives, however, did not show any synergistic effect on the intestinal morphology.