Harry Kane claimed the referee "bottled it" after England were robbed of the stoppage-time goal that would have prevented their UEFA Nations League opener ending in defeat to Spain.
Back in action for the first time since equalling their best-ever World Cup performance on foreign soil, the Three Lions got off to a superb start as Marcus Rashford swept home Luke Shaw's exceptional pass.
But Saul Niguez and Rodrigo turned the match on its head as Spain ran out 2-1 victors on a night when Shaw's nasty-looking injury early in the second half took the sting out of proceedings.
England rallied late on - perhaps buoyed by positive news from the dressing room about the left-back's condition - and looked to have snatched a draw through substitute Danny Welbeck.
But referee Danny Makkelie infuriated the hosts by ruling that the forward had fouled David De Gea when collecting a high ball, leading to sharp words from captain Kane.
Asked if the goal should have stood, the England striker told Sky Sports Football: "Yeah, 100 per cent.
"In those big environments you need the ref to stay strong and unfortunately he's bottled it.
"Danny Welbeck just stood there, the keeper went to catch it, he's put it away - I don't know how it's a foul.
"You need a firm ref who under the pressure doesn't give wrong decisions.
"Disappointing to finish that way, I thought we played well - especially second half - and deserved a draw at least.
"It was a good game, it was a real test against a team that keep the ball really well. We probably created the better chances throughout the game."
Southgate echoed his captain's frustration but did not allow the official's mistake to mask some of England's shortcomings.
"I think the two defenders tried to block Danny out of it," he said. "De Gea came over the top and just dropped it.
"I think it's clear for everybody to see that it should have been a goal.
"But over the 90 minutes we have to accept that Spain were better than us for long periods of the game.
"We know the quality they possess. Their retention of the ball was top class.
"We were a little bit disjointed in some of our pressing, particularly in the first half.
"But even when you press well against them, they have some wonderful players that can wriggle out of problems and play first time out of situations.
"At the moment, we're not able to do that when teams are pressing us high.
"As the game wore on, we started to work that out and I thought Eric (Dier) provided some stability for us, and him and John (Stones) being able to rotate positions allowed us to play through midfield better.
"I'm very proud of the way the players finished the game because it was a night when you know you are playing against a top team.
"I've seen teams fold in that sort of situation, but we finished really strongly, created a couple of very good chances, have probably had as many shots on target as they have.
"So, given where we're at and we need those experiences to improve, I couldn't ask any more of the players."
Recently-appointed Spain head coach Luis Enrique was not as forthcoming about the disallowed Welbeck goal, but believes the result was deserved.
"I couldn't see the play from my place," he said after the Group A4 encounter. "It was difficult.
"I asked David De Gea and he told me he was fouled, but I don't know. I cannot swear.
"We have to think about the whole match and I think we deserved to win."