The Barcelona midfielder was incensed as Sergio Ramos scored the home side's second goal, only for the man in the middle to blow for full time.
By then nearly 98 minutes were on the clock, after seven minutes of injury time had been added by the fourth official for lengthy pauses during the second half.
And after Ramos nodded Spain back into it, the referee blew there and then for full time, before they could run back to the centre circle and take the kick-off.
Spain were furious with the decision, but time was up.
And Busquets, 30, appeared to launch the ball at the Polish referee, before apologising, according to Marca.
Whether it was to throw it at him, to simply give the ball back, or an act of pure petulance, Busquets will be investigated if it is in the referee's notes to Uefa.
Alvaro Morata was booked for appearing to swear at the official, as he was surrounded by furious Spanish players.
And even Busquets' team-mates appeared to have to duck out of the way of the ball as it flew past.
Many fans thought the Barcelona midfielder should have been booked for a tactical foul on Raheem Sterling as he prevented the Manchester City ace getting a flying counter attack going.
Spain went 3-0 down thanks to two goals from Sterling and one from Marcus Rashford, before Paco Alcacer's header from a corner got them back to 3-1 on 58 minutes.
It was backs to the wall then for England, who switched to a three-man central defence and weathered the storm until the 98th minute.
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