Streptococcus agalactiae, referred to as Group B Streptococcus (GBS), is a prominent bacterium causing life-threatening neonatal infections. Although antibiotics are efficient against GBS, growing antibiotic resistance forces the search for alternative treatments and/or prevention approaches. Antimicrobial photodynamic inactivation (aPDI) appears to be a potent alternative non-antibiotic strategy against GBS. The effect of rose bengal aPDI on various GBS serotypes, Lactobacillus species, human eukaryotic cell lines and microbial vaginal flora composition was evaluated. RB-mediated aPDI was evidenced to exert high bactericidal efficacy towards S. agalactiae in vitro (>4 log10 units of viability reduction for planktonic and >2 log10 units for multispecies biofilm culture) and in vivo (ca. 2 log10 units of viability reduction in mice vaginal GBS colonization model) in microbiological and metagenomic analyses. At the same time, RB-mediated aPDI was evidenced to be not mutagenic and safe for human vaginal cells, as well as capable of maintaining the balance and viability of vaginal microbial flora. aPDI can efficiently kill GBS and serve as an alternative approach against GBS vaginal colonization and/or infections.