Abstract: Nervous necrosis virus (NNV) is a highly pathogenic agent that threatens global aquaculture. Berberine (BBR) is a natural alkaloid with broad-spectrum antiviral activity, yet its effect and mechanism against NNV remain unclear. This study investigated the antiviral effect and mechanism of BBR against NNV in vitro. The safe concentration of BBR was determined via CCK-8 assay. Antiviral activity was assessed through cytopathic observation and RT-qPCR detection of NNV capsid protein gene expression. Mechanistic insights were gained through virus pretreatment, time-of-addition assays, RT-qPCR, and flow cytometry, evaluating BBR’s direct virucidal effect and its interference with viral adsorption, entry, and intracellular replication. Additionally, RT-qPCR was used to examine BBR’s modulation of NNV-induced expression of pro-inflammatory cytokines IL-1β and IL-8. Results showed that BBR significantly inhibited NNV proliferation at non-cytotoxic concentrations. Mechanistically, BBR directly inactivated viral particles, blocked viral adsorption and cellular entry, and suppressed intracellular replication. It also downregulated NNV-induced overexpression of IL-1β and IL-8. In conclusion, BBR exerts anti-NNV effects through multi-target actions, including direct antiviral activity, inhibition of key infection steps, and attenuation of host inflammatory response. These findings provide a theoretical foundation for developing BBR as a green antiviral agent against NNV in aquaculture.