The experienced 32-year-old suffered a hyperextended right knee in Ecuador's 3-1 defeat to Mexico on the weekend in a sickening clash with Luis Montes, who broke his leg in the collision.
Castillo, who plays his club football for Saudi side Al-Hilal, is set for further tests on Monday to ascertain the severity of the injury.
Despite his dream of featuring in Brazil being in jeopardy, Castillo is remaining hopeful he can participate at football's premier event.
"You have to be calm and have faith," he told the Ecuador Football Federation's official website. "I'm hoping it's not something serious to allow me to be at the World Cup.
"I am going to do tests to determine where the injury is at."
Ecuador face England in Miami on Wednesday in their final World Cup warm-up fixture, before their Group E opener with Switzerland on June 15.
