Vaccines prepared from formalin-killed Streptococcus agalactiae were administered to Nile tilapia (Oreochromis niloticus) via three different routes: immersion in a water-based vaccine, injection with an oil-based vaccine, and as a water-based oral vaccine. All vaccination treatments increased lysozyme and peroxidase activity in skin mucus of Nile tilapia by 1.2- to 1.5-fold compared to their activities in unvaccinated control fish. Likewise, alternative complement, phagocytosis, and respiratory burst activities in the blood serum of the vaccinated fish were 1.2- to 1.5-times higher than in the unvaccinated fish. In addition, the expression transcripts of interleukin-1 (IL-1), interleukin-8 (IL-8), and lipopolysaccharide-binding protein (LBP) were 2.3- to 2.9-fold higher in the vaccinated fish compared to those in the unvaccinated control. The unvaccinated fish challenged with Streptococcus agalactiae had a survival rate of 25% compared to a survival rate of 78–85% for the vaccinated fish. The differences between the unvaccinated and vaccinated fish were all statistically significant, but there was no significant difference in any of the indicators of immunity between the three vaccinated groups. Collectively, these results confirm that vaccination with formalin-killed Streptococcus agalactiae significantly improved the resistance of Nile tilapia to infection by the pathogen. Overall, the efficacy of oral administration of the vaccine was comparable to that of vaccine administered via injection, indicating that oral vaccination is a viable cost-effective alternative to administering vaccines by injection.