Streptococcus agalactiae is a highly invasive bacterium that causes significant economic losses in tilapia aquaculture around the world. Furthermore, it is a pathogen for mammals, including humans, emphasizing its importance in One Health. The aim of this work was to evaluate the evolution of clinical and histopathological lesions caused by acute infection with two serotypes of S. agalactiae. For this, two strains isolated from natural outbreaks in Brazilian aquaculture farms (S13, serotype Ib; S73, serotype III) were used to challenge juvenile Nile tilapia (Oreochromis niloticus) intraperitoneally. Target organ samples were collected ten times, between 1 and 96 h post-infection, for microbiological and histopathological analyses. Anorexia was the first clinical sign and the first death occurred at 24 and 30 h in the fish infected with strains S13 and S73, respectively. Serotype Ib initially caused more pronounced lesions in the nervous system; however, serotype III lesions progressed more aggressively, reaching the same severity as those of serotype Ib. This trend was repeated in the mortality curve after 32 h. These results elucidated the important stages in the pathogenesis of S. agalactiae serotypes Ib and III in tilapia and suggest “tips and tricks” to improve the positive culture rate in the clinical diagnosis of infections in some tissues.