Three people have already died of the deadly pathogen in Seville. Now the virus has claimed its first victim in the neighbouring province of Cadiz. On Monday an 87-year-old woman died in Puerto Real Hospital in the province of Cadiz.
Authorities said there are another five cases in the same province.
The virus is passed via mosquito bite.
Spanish authorities have outlined West Nile Virus risk areas in the southern regions of Cadiz and Puerto Real.
Seville is also a risk area with a number of confirmed cases there standing at 24.
The three fatalities in Seville were two men aged 70 and 77 and a woman aged 85.
Infectious disease expert Pablo Barreiro said: “Only about two to five percent of the cases present symptoms.
“It’s an illness that can go unnoticed very easily.”
He advised applying insect repellent as the most effective solution.
READ MORE: Spain panic: Seville reports first death from West Nile virus
However, it can cause meningitis in the brain in those with compromised immune systems.
Encephalitis, inflammation of the brain, will happen in less than 1 percent of infected cases.
As of last Wednesday, there have been 66 cases of the virus reported across the EU.
Greece has had the largest outbreak, there have also been cases diagnosed in Italy and Romania.