The hybrid marbled goby (Oxyeleotris marmoratus ♀ × Oxyeleotris lineolatus ♂) is a high-value aquaculture species in China, with rapidly increasing production in recent years. While Streptococcus agalactiae is a well-known pathogen in various fish species, its association with marbled goby has not been previously reported. Here, we present the first confirmed case of S. agalactiae infection in farmed hybrid marbled goby. Infected fish exhibited typical signs of streptococcosis, including erratic swimming, hemorrhages at fin bases, skin ulcers, and cloudy or hemorrhagic eyes. A Gram-positive bacterial strain, designated ZH2208, was isolated from diseased fish. Morphological, biochemical, 16 S rRNA sequencing, and phylogenetic analyses identified the isolate as S. agalactiae, serotype Ia-a dominant serotype previously associated with tilapia infections in Guangdong, China. Challenge experiments using doses of 1 × 10⁴ and 1 × 10⁷ CFU/fish confirmed the high virulence of ZH2208, resulting in 50% and 100% mortality, respectively. Eleven virulence genes involved in host invasion, hemolysis, immune evasion, and blood-brain barrier penetration were detected by PCR. Histopathology revealed typical lesions in gills, liver, spleen, kidney, and brain, including acute inflammation, hemorrhage, and neuronal degeneration. Notably, pronounced swim bladder swelling with distinct hemorrhagic spots was observed. Antibiotic susceptibility testing showed that ZH2208 was sensitive to enrofloxacin and doxycycline but resistant to several common aquaculture antibiotics, highlighting its multidrug-resistant profile. In conclusion, this study provides the first comprehensive characterization of S. agalactiae infection in hybrid marbled goby. These findings support the development of effective prevention and control strategies, including vaccination and rational antibiotic use, to manage streptococcal disease in goby aquaculture.