"Staging a referendum is a mistake, we have opened a Pandora's box," Guindos told a business forum late Monday.
"I am convinced Brexit will not happen, I am convinced that the British will be rational," he added.
"Please let's not get into this situation again, because it can have effects in other nations where populism is advancing very quickly."
De Guindos said the decision to call the referendum has "a component of populism."
If Britain votes to leave the EU it would be "a political failure" for British Prime Minister David Cameron's conservative government, he said.
His comments come as polls suggest the outcome in Thursday's vote could go either way. Analysts have predicted a Brexit could unleash a domino effect within the 28-nation bloc.