Microglial activation is a key driver of neuroinflammation, rendering it a critical therapeutic target. This study explored the neuroprotective potential of compounds derived from Swertia mussotii Franch. against lipopolysaccharide (LPS)-induced microglial activation. Eleven compounds were isolated from this plant. Screening using an LPS-stimulated BV2 microglial cell model identified amarogentin (AMG) as exhibiting significant protective effects. Subsequent mechanistic investigations revealed that AMG effectively mitigated LPS-induced morphological changes associated with microglial activation and suppressed the release of nitric oxide (NO) (p < 0.01) and pro-inflammatory cytokines (TNF-α, IL-1β) (p < 0.05). Moreover, AMG markedly downregulated the mRNA expression of inducible nitric oxide synthase (iNOS) (p < 0.01), TNF-α (p < 0.01), and IL-1β (p < 0.05). At the protein level, AMG inhibited the expression of phosphorylated p65 (p-p65) (p < 0.01). These results indicate that AMG confers robust neuroprotective effects against microglial activation-induced injury, likely via suppression of the NF-κB signalling pathway.