Background: West Nile virus (WNV) is a zoonotic mosquito–transmitted arbo-virus belonging to the genus flavivirus in the family Flaviviridae. Objective: The aim of the present study was to determine the role of WNV in human cases with fever of unknown etiology and to study the immune status to WNV among healthy humans. In addition, the prevalence of WNV in Culex mosquito vectors was estimated. Methods: The study included 210 patients (with undiagnosed acute febrile illness for more than 2 days; admitted to Alexandria Fever Hospital during a period of one year), 200 healthy humans (volunteer blood donors accepted for donation at Alexandria Regional Blood Bank) and 200 mosquito pools (from six administrative districts of Alexandria). WNV antibodies of the IgM and IgG types were detected in patients and healthy humans using enzyme linked immunosorbent assay (ELISA), respectively. WNV was detected in mosquito by virus isolation on Vero cell culture. Results: WNV IgM antibodies were detected in 56 (26.7%) patients, while WNV IgG antibodies were detected in 42 (21%) out of the 200 healthy humans. WNV was isolated from 27 (13.5%) out of the 200 mosquito pools. Patients in the age groups 20- and 40- had the highest percentages of WNV IgM antibodies. The highest WNV IgM antibodies positivity rates occurred in spring and in fall seasons. Male samples had significantly higher percentage of the IgG antibodies than female samples (25.6%Vs 13.3%). Conclusion: WNV should not be overlooked in diagnosis of fevers as the main problem may be not due to the disease itself but the economic consequences from the hospitalization of indeclinable numbers of patients. WN disease can be difficult to monitor; almost unpredictable and appearing unexpectedly, this disease has to be surveyed and the viral circulation rapidly detected so that the sanitary authorities can take protective measures.