This study reports a unique case of West Nile Virus (WNV)-associated multilevel macular ischemia, involving retinal capillary plexuses and choriocapillaris, confirmed through real-time polymerase chain reaction (RT-PCR) of aqueous humor and characterized using advanced multimodal imaging. A 35-year-old immunocompromised male presented with bilateral, acute, and painless visual loss persisting for 14 days. Ophthalmic evaluation included ultra-widefield fundus photography, spectral-domain optical coherence tomography (SD-OCT), OCT angiography (OCT-A), fluorescein angiography (FA), and indocyanine green angiography (ICGA). Laboratory testing included serology and RT-PCR analysis of aqueous humor. Presenting visual acuity was hand motion in both eyes. Fundus examination revealed bilateral pale optic discs, attenuated arteries, and deep creamy chorioretinal lesions in the posterior pole and mid-periphery. SD-OCT demonstrated nerve fiber layer ischemia and hyperreflective bands in the inner and outer nuclear layers. OCT-A showed flow deficits in the deep capillary plexus and choriocapillaris. FA and ICGA revealed active lesions with late-phase leakage and hyperfluorescence, alongside target-like patterns suggesting chronic atrophy. RT-PCR analysis of aqueous humor confirmed WNV infection with a viral load of 1,335 copies/mL. Serial imaging demonstrated the progression to inner retinal and RPE atrophy over 8 weeks. This case represents the first documented use of RT-PCR analysis of aqueous humor to confirm WNV infection, providing a novel diagnostic approach for atypical presentations. Multimodal imaging, including SD-OCT and OCT-A, revealed multilevel ischemic retinal damage and highlighted the acute and chronic lesions. These findings underscore the importance of advanced imaging and molecular diagnostics in identifying and managing ocular manifestations of WNV.