Spain is considering a 50 euro (£43) fee to cross its border with Gibraltar, amid a row over an artificial reef.
Foreign Minister Jose Manuel Garcia-Margallo told a Spanish newspaper the proceeds would "help fishermen affected by the destruction of fishing grounds".
The latest tensions come after the British territory began work on the concrete reef, which Spain claims infringes the rights of its fishermen.
The UK Foreign Office said it was "concerned" at the minister's comments.
Britain has governed Gibraltar for 300 years but Spain disputes UK sovereignty over the rocky outcrop on its southern tip.
In the interview with ABC newspaper, published on Sunday, Mr Garcia-Margallo said Spain was considering the fee to enter and exit Gibraltar through its border post with Spain.
The minister was also quoted as saying that Spanish tax authorities could launch an investigation into property owned by around 6,000 Gibraltarians in neighbouring parts of Spain.
Spain is also considering closing its airspace to flights heading to Gibraltar, and changing the law so that online gaming companies operating from the British overseas territory have to use Spanish servers and come under the jurisdiction of Madrid's taxation regime, he said.
He cited the "extreme measure" by Gibraltar of "throwing concrete blocks with spikes that destroy the fishing grounds" - thought to be in reference to the reef.
He said that as well as the border fees measure, Spain would stop concrete and other materials being brought in through the border for the building of the reef.
A spokeswoman for the UK Foreign Office said: "We are concerned by today's comments on Gibraltar, which we are looking into further.
"As we have said, we will not compromise on our sovereignty over Gibraltar, nor our commitment to its people. We continue to use all necessary measures to safeguard British sovereignty."
Gibraltar resident Amber Lyons, 21, said thousands of people who work across the border in Spain would be affected if they had to pay to cross over.
"My boyfriend lives just over the border so I would have to pay 100 euros each time I wanted to see him," said Ms Lyons, who works for the border and coastguard agency.
Ms Lyons, whose grandparents also live close to the Spanish side of the border, added: "I didn't really believe it when I heard it - surely it would contravene our human rights."
Gibraltar started work on the artificial reef by placing concrete blocks in the sea 10 days ago.
Spain lodged a complaint with the UK over the reef which it said stopped "Spanish fishermen fishing in a manner that is contrary to our law".
Spanish authorities later increased vehicle searches, resulting in three days of delays at the Gibraltar-Spain border last weekend.
Gibraltar linked the delays to Spain's anger over the reef - but Spain denied this, saying it had a duty to prevent smuggling.
The Foreign Office said the delays stopped on Monday after Foreign Secretary William Hague called his counterpart in Madrid.
On Friday Spain's ambassador Federico Trillo was summoned to give assurances there would be no repeat.