During the summer-autumn of 2011, a human outbreak of West Nile virus (WNV) infection occurred in southern Greece, following the first outbreak during 2010 in northern Greece. An investigation was performed to analyze laboratory diagnosis, geographic distribution, and clinical features of WNV cases in southern Greece. Serum and cerebrospinal fluid (CSF) specimens from all patients seeking laboratory diagnosis for suspected WNV infection were tested for the presence of specific WNV immunoglobulin M (IgM) and IgG antibodies. Detection of WNV RNA in CSF and whole blood samples was accomplished by real-time PCR. During August-October of 2011, 31 confirmed or probable cases of WNV infection were identified. In 25 of them, individuals experienced severe neurological manifestations and were classified as WNV neuroinvasive disease cases. Risk factors such as advanced age, hypertension, and diabetes mellitus were identified in most cases with neurological complications. As many as 25 of the WNV cases occurred in the broader region of Athens; the majority of them (17 cases) were identified in municipalities of Eastern Attica, located almost 40 km from the metropolitan area of Athens and 500 km from Central Macedonia, where the 2010 WNV outbreak occurred. The spread of the virus in a newly affected area of the country suggests that WNV has been established in Greece and disease transmission will be continued in the future.