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An experiment was conducted to assess the response of chicks to in-ovo injection of Bacillus subtilis(probiotic), raffinose (prebiotic), and their combinations. The study used 1,500 embryonated eggs allotted to 10 groups/ 6 replicates (150 eggs/group). The experimental treatments were: 1) un-injected control (NC); 2) sham (sterile distilled water) (PC); 3) probiotic 4 × 105CFU/egg (LBS); 4) probiotic 4 × 106CFU/egg (HBS); 5) prebiotic 2 mg/egg (LR); (6 prebiotic 3 mg/egg (HR); 7) probiotic 4 × 105CFU + prebiotic 2 mg/egg (LBS+LR); 8) probiotic 4 × 105CFU + prebiotic 3 mg/egg (LBS+HR); 9) probiotic 4 × 106CFU + prebiotic 2 mg/egg (HBS+LR); and 10) probiotic 4 × 106CFU + prebiotic 3 mg/egg (HBS+HR). Results showed that in-ovo inclusion of Bacillus subtilis, prebiotic, and their combinations improved hatchability, yolk-free chick weight, and chick weight compared to the control group. Moreover, the in-ovo treatment reduced residual yolk weight on the day of hatch compared to the control group. Different levels of in-ovo B. subtilisalone or combined with raffinose significantly (P≤ 0.001) reduced total bacterial count and total yeast and mold count compared to the negative control group. Total coliform and E. colidecreased significantly (P≤ 0.001) in groups treated with probiotics, prebiotics, and synbiotics with different doses during incubation compared to those in the control. Clostridiumspp. was not detected in the groups injected with B. subtilisalone or combined with raffinose. In-ovo probiotics and synbiotics (LBS+LR & LBS+HR) significantly (P≤ 0.001) increased ileal villus length compared to other groups. In-ovo treatment increased mRNA expression of JAM-2compared to the control group. The fold change significantly increased in group LBS+HR for genes MUC-2, OCLN, VEGF, SGLT-1, and EAAT-3compared to the negative control. In conclusion, in-ovo injection of a low dose of B. subtilisplus a high or low dose of raffinose can positively affect hatching traits, cecal microbial populations, intestinal histomorphometry, nutrient transport- and intestinal function-related genes, and chick quality of newly hatched broiler chicks.