Human cytomegalovirus (HCMV) causes severe diseases (e.g., retinitis, pneumonitis) in immunocompromised individuals, but current anti-HCMV agents suffer from toxicities, poor oral bioavailability, and emerging drug resistance. As part of our antiviral research, we screened in-house and external compounds and identified 1,6-naphthyridine 1 as an HCMV inhibitor with potency and selectivity similar to ganciclovir. We then designed and synthesized analogues of 1, optimized the amine portion and naphthyridine substituents, and conducted preliminary structure-activity relationship (SAR) studies. SAR results demonstrated that an ortho-isopropoxy group on the benzylamine and substitution at the naphthyridine C-8 position are highly beneficial for potency and selectivity. Compounds 10 (vinyl-substituted, IC50 = 3 ng/mL, selectivity index >5000), 11 (methyl-substituted, IC50 = 0.4 ng/mL, selectivity index >7000), and 17 (ortho-isopropoxy, IC50 = 5 ng/mL, selectivity index = 500) showed exceptional potency and selectivity. Notably, these inhibitors were not cross-resistant to ganciclovir or cidofovir, suggesting a unique mode of action. In conclusion, we have discovered a novel class of 1,6-naphthyridine-based HCMV inhibitors, defined their structural requirements for activity, and identified several highly potent and selective analogues. Ongoing studies will further explore their mechanisms and pharmacokinetics.