Abstract: This study aimed to investigate the antibacterial activity and underlying mechanism of Gardenia jasminoides essential oil (GEO) against Streptococcus agalactiae (GBS). GEO was extracted using supercritical CO₂ fluid extraction, and its chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS). Results showed that GEO was primarily composed of alcoholic and olefinic compounds. In vitro assays revealed that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of GEO against GBS were 3.13 and 12.50 mg/mL, respectively. GEO at 1/2 MIC significantly inhibited bacterial growth, disrupted cell membrane integrity, and led to increased protein leakage and alkaline phosphatase activity (P<0.05). Observations via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that GEO at 1/2 MIC induced ultrastructural damage, including membrane rupture, cytoplasmic leakage, and vacuolization. Moreover, GEO at 1/2 MIC markedly suppressed biofilm formation and reduced the production of the quorum-sensing signal molecule autoinducer-2 (AI-2). In vivo challenge tests demonstrated that dietary supplementation with GEO (2g/kg) significantly enhanced the survival rate of Acrossocheilus fasciatus upon GBS infection and notably reduced the bacterial load in the liver and brain tissues. These findings suggest that GEO possesses notable antibacterial activity against GBS, likely through the disruption of cellular structures, and exhibits potential for development as a green antibacterial agent in aquaculture.