In Andalusia, Spain, West Nile virus (WNV) surveillance takes place from April to November, during the active vector period. Within this area seroconversion to this virus was evidenced in wild birds in 2004, affecting horses and two humans for the first time in 2010. Since 2010, the virus has been isolated every year in horses, and national and regional surveillance plans have been updated with the epidemiological changes found. WNV is spreading rapidly throughout southern Europe and has caused outbreaks in humans. Here we describe the second WNV outbreak in humans in Andalusia, with three confirmed cases, which occurred between August and September 2016, and the measures carried out to control it. Surveillance during the transmission season is essential to monitor and ensure prompt identification of any outbreaks.