Modric was withdrawn in the second half against the Czech Republic with Croatia leading 2-0 and cruising towards the last 16, before the Czechs rallied to claim an unlikely draw.
"Modric's groin is strained and swollen," team doctor Boris Nemec said on Saturday.
"He will train at less than full intensity in the next few days and is very unlikely to be available for the Spain match," he said.
Euro 2016
Rooney: Hodgson got it wrong at finals
01/08/2016 AT 19:37
Modric, who struck the winner when Croatia beat Turkey in their opening game, has been in imperious form so far at Euro 2016, and Ante Cacic will likely now turn to Real Madrid's Mateo Kovacic to plug the hole in his midfield.
Vedran Corluka is also a doubt after the stitches he received in a head wound sustained against Turkey forced him to wear a water polo cap against the Czechs.
The Czech's late leveller has placed added pressure on Croatia for their final group game against the holders, with Cacic's side needing a positive result to keep qualification for the last 16 in their own hands.
Euro 2016
Hodgson's England 'had the talent to win Euro 2016'
26/07/2016 AT 15:39
Euro 2016
France beat Portugal to make final
21/07/2016 AT 17:19