Acanthamoeba spp., pose a growing global health concern due to their association with granulomatous amoebic encephalitis (GAE), particularly in immunocompromised individuals. Treatment options remain limited and often ineffective due to drug resistance, delayed diagnosis, and the parasite’s ability to induce neuroinflammation and disrupt the blood-brain barrier (BBB). Therefore, this study investigates, for the first time, the therapeutic role of quercetin-conjugated silver nanoparticles (Q-AgNPs) in modulating neuroinflammatory responses, enhancing endothelial barrier integrity, and improving pharmacokinetic properties of this nanoformulation in an experimental model. A total of 105 male mice were divided into seven groups, representing both immunocompetent and immunosuppressed animals, with appropriate control groups. Q-AgNPs were synthesized and characterized using spectrophotometer, transmission electron microscope, zeta-potential, loading efficiency, invitro release analysis, and FTIR analysis. Hematological indices showed improvement over time with treatment, with significant increases in erythrocytes, hemoglobin, and hematocrit levels, alongside gradual reduction in humoral inflammatory reaction. Additionally, Q-AgNPs treatment reduced proinflammatory cytokines (TNF-α and IL-1β) and BBB permeability marker (MMP9), while significantly increasing anti-inflammatory cytokine (IL-10). Histopathological examinations showed that Q-AgNPs alleviated neural lesions and glial cell reaction. Immunohistochemical analysis highlighted the role of Q-AgNPs in reducing the trans-endothelial immune cell migration and in delaying GAE-induced brain atrophy through decreased PECAM-1 gene expression in the brain regions. Molecular docking revealed that Q-AgNPs had a strong binding affinity with the mitochondrial respiratory protein NAD2, offering enhanced pharmacokinetic properties compared to quercetin alone. This study support Q-AgNPs as a promising therapeutic strategy to combat A. polyphaga-induced GAE by modulating neuroinflammation pathways and improving drug delivery.