A 77-year old man became the first victim on Thursday after being admitted to hospital in Seville while an 85-year-old woman succumbed on Friday.
A total of 35 people have so far been confirmed to be infected in the outbreak which has hit the areas of Coroa del Rio and La Puebla del Rio, two communities on the banks of the Guadalquiver River outside Seville.
Of these, 23 people have been hospitalised and seven are being treated in intensive care units.
The virus is transmitted by the common Culex mosquito and can causes inflammation of the brain and in rare cases death.
Residents in the affected areas have been warned to be vigilant and avoid mosquitos by covering up and using repellents while authorities have been fumigating problem areas.