The Manchester United forward and Southampton defender both missed today's training with "minor problems", according to Sky Sports.
England skipper Rooney has started all but one of Gareth Southgate's games in charge of England and may be replaced by Jamie Vardy for the Spain match.
Bertrand started Southgate's first game in charge of England against Malta but was understudy to Danny Rose in England's 3-0 victory over Scotland.
Should Bertrand miss the match against Spain, Danny Rose may continue at left-back or Southgate could call on Aaron Cresswell who is yet to make his England debut.