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Recent studies have highlighted the potential of unsaturated fatty acids in natural oils to influence virus replication. This study extracted ginseng seed oil (GSO) and evaluated its physiochemical parameters, lipid class, and oxidation stability. GSO was then synthesized into an oil/water nanoemulsion (NE) through optimized formulation, time, temperature, and storage conditions. GSO-NE showed no cytotoxicity up to 5 µg/mL but effectively inhibited HCoV-OC43 replication and suppressed proinflammatory biomarkers in infected cells. RNA sequencing revealed significant alterations in the gene expression profile by GSO-NE treatment, particularly implicating the ABC transporters signaling pathway. Notably, GSO-NE downregulated ABCG subfamily membrane proteins, crucial for lipid regulation in virus-infected cells. The downregulation of these genes (ABCG1, ABCG2, ABCG4, and ABCG5/8) was reversed by GSO-NE, as demonstrated through qRT-PCR. Furthermore, the downregulation of ABCG1 and ABCG5/8 genes was confirmed using immunoblotting techniques. These findings suggest GSO-NE as a promising avenue for future research or therapeutic applications.