Alex McCarthy and Danny Rose have both withdrawn from the England squad and returned to their respective clubs after failing to recover from minor injuries.
According to England's Twitter account, both players arrived at the training camp with knocks and the decision has been made that neither will be risked.
Southampton goalkeeper McCarthy, who is third in the pecking order behind Jordan Pickford and Jack Butland, would have been unlikely to play in either of England's matches anyway. He received his first call-up last month, but is still awaiting his debut.
The injury to Tottenham left back Rose is more of a blow for Gareth Southgate. Rose was the best player in England's last match against Switzerland. With Luke Shaw also out injured, Ben Chilwell could make his full debut on Friday.
It has not yet been revealed if any replacements will be drafted in to replace McCarthy and Rose. Lewis Dunk was brought into the squad earlier this week after James Tarkowski withdrew with an injury.
England face Croatia behind closed doors in Rijeka on Friday, before making the trip to Seville for a meeting with Spain four days later.
The Three Lions lost their first Nations League match last month to Spain, who top the group with six points after two games.