Abstract: This study aimed to evaluate the therapeutic efficacy and mechanism of enrofloxacin nanoemulsion (ENR-NE) against streptococcal meningitis in tilapia (Oreochromis niloticus). A tilapia infection model was established by intraperitoneal injection of 1.5×10⁸ CFU Streptococcus agalactiae. A total of 240healthy Nile tilapia was randomly allocated into four treatment groups: control group, S. agalactiae infection group, enrofloxacin powder treatment group (ENR group), and enrofloxacin nanoemulsion treatment group (ENR-NE group). At 12h post-infection, fish in the two treatment groups received a daily oral dose of 10 mg/kg body weight of ENR or ENR-NE for 3 consecutive days. Survival rates, drug accumulation, histopathological changes, and related gene expression were subsequently evaluated. The results showed that the cumulative survival rate in the ENR-NE group (75%) was significantly higher than that in the S. agalactiae group (10%) and the ENR group (40%). Tissue drug concentration analysis revealed that the enrofloxacin concentration in the brain of the ENR-NE group remained at a relatively high level at 48h after the last administration, which was significantly higher than that in the ENR group (P<0.05), confirming that the nanoemulsion formulation effectively enhances the drug’s ability to penetrate the blood-brain barrier (BBB). Furthermore, ENR-NE significantly alleviated inflammatory cell infiltration in the brain, mitigated the abnormal increase in splenic Melano-macrophage centers (MMCs), and restored serum alanine aminotransferase (ALT) activity to normal levels. Both ENR and ENR-NE effectively reversed S. agalactiae-induced hepatic oxidative stress damage, with no significant differences observed between the two groups in terms of superoxide dismutase (SOD), catalase (CAT) activities, or malondialdehyde (MDA) content. Molecular biological analyses revealed that ENR-NE significantly upregulated the mRNA expression levels of tight junction protein genes (claudin-5, zo-1) in the brain (P<0.05), while downregulating the expression of nerve injury-related markers (gfap, s100) (P<0.05). Moreover, ENR-NE was significantly superior to the ENR group in restoring the transcriptional levels of pattern recognition receptors (nod1, nod2) and pro-inflammatory cytokines (tnf-α, il-1β) in the liver and spleen (P<0.05). In conclusion, ENR-NE significantly enhances the therapeutic efficacy against streptococcal meningitis in tilapia through mechanisms involving improved cerebral drug accumulation, preservation of BBB integrity, inhibition of neuroinflammatory responses, and regulation of systemic immune homeostasis. These findings provide experimental evidence supporting the development and application of ENR-NE as a novel antibiotic formulation for aquaculture.