Banning antibiotic growth promoters has negatively impacted poultry production and sustainability, which led to exploring efficient alternatives such as probiotics, probiotics, and synbiotics. Effect of in ovo injection of Bacillus subtilis, raffinose, and their synbiotics on growth performance, cecal microbial population and volatile fatty acid concentration, ileal histomorphology, and ileal gene expression was investigated in broilers (Gallus gallus) raised for 21 days. On 300 h of incubation, a total of 1,500 embryonated eggs were equally allotted into 10 groups. The first was non-injected (NC) and the remaining in ovo injected with sterile distilled water (PC), B. subtilis 4 × 105 and 4 × 106 CFU (BS1 and BS2), Raffinose 2 and 3 mg (R1 and R2), B. subtilis 4 × 105 CFU + raffinose 2 mg (BS1R1), B. subtilis 4 × 105 CFU + raffinose 3 mg (BS1R2), B. subtilis 4 × 106 CFU + raffinose 2 mg (BS2R1), and B. subtilis 4 × 106 CFU + raffinose 3 mg (BS2R2). At hatch, 60 chicks from each group were randomly chosen, divided into groups of 6 replicates (10 birds/replicate), and fed with a corn-soybean-based diet. In ovo inoculation of B. subtilis and raffinose alone or combinations significantly improved body weight, feed intake, and feed conversion ratio of 21-day-old broilers compared to NC. Cecal concentrations of butyric, pentanoic, propionic, and isobutyric acids were significantly elevated in R1, R2, BS2R1, and BS2R2, whereas isovaleric and acetic acids were significantly increased in R1 and BS2R1 compared to NC. Cecal microbial population was significantly altered in treated groups. Ileal villus height was increased (p < 0.001) in BS1, R2, and BS2R2 compared to NC. The mRNA expression of mucin-2 was upregulated (p < 0.05) in synbiotic groups except for BS1R1. Vascular endothelial growth factor (VEGF) expression was increased (p < 0.05) in BS2, R1, BS1R1, and BS1R2 compared to NC. SGLT-1 expression was upregulated (p < 0.05) in all treated birds except those of R1 group compared to NC. The mRNA expressions of interleukin (IL)-2 and toll-like receptor (TLR)-4 were downregulated (p < 0.05) in BS2 and R1 for IL-2 and BS1R1 and BS2R2 for TLR-4. It was concluded that in ovo B. subtilis, raffinose, and synbiotics positively affected growth performance, cecal microbiota, gut health, immune responses, and thus the sustainability of production in 21-day-old broilers.