Juanfran has been diagnosed with a hamstring problem, adding to Atletico Madrid's injury issues.
Fellow full-back Filipe Luis fractured his fibula in Thursday's Europa League defeat of Lokomotiv Moscow and is expected to miss the World Cup.
While Juanfran's injury is less serious, it adds to Atletico's issues after the January departures of Yannick Carrasco, Nicolas Gaitan and Miguel Angel Moya reduced Diego Simeone's options.
"Juanfran was taken off during half-time in our Europa League match against Lokomotiv Moscow due to some muscular discomfort," Atletico said in a statement.
"He underwent medical exams in Madrid and was diagnosed with an injury to his left hamstring."
Medical exams can confirm has fractured his left fibula. Here’s to a speedy recovery! March 16, 2018
Juanfran will miss Sunday's LaLiga trip to Villarreal and could be out of next month's Europa League quarter-final against Sporting CP, as well as a derby clash with rivals Real Madrid in the league.
The 33-year-old has started 10 LaLiga games this season, battling for selection at right-back with Sime Vrsaljko, while he last played for Spain in 2016.
Atletico are second in the LaLiga table, eight points behind leaders Barcelona but seven clear of third-placed Madrid.
