Supporters in the Benito Villamarin stadium in Seville began a chorus of boos prior to the Nations League fixture that saw England win 3-2.
It was a first competitive home defeat for Spain in 15 years.
News of possible sanctions comes just 24 hours after hooligans were involved in a rampage through the streets of Seville.
Riot police intervened when England fans began vandalising cars during a boozy night before the game.
Footage showed noisy supporters kicking wing mirrors off vehicles and circling drivers in their cars.
But it looks set to be the Spanish who are punished for their whistling, especially as it's not the first time.
Spain were fined after the 2010 European Championships when fans booed during the Irish national anthem.
An announcement was made at the stadium on Monday night asking fans to respect the English anthem.
But the warnings were not heeded.
It will be up to Uefa's delegate report on the severity of the fine but Spain fear they could be forced to play behind closed doors if it continues.
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