The fishermen say the reef restricts their right to fish, but Gibraltar says they should not be fishing there, BBC reported.
Gibraltar police said the protest, which lasted an hour and passed off without incident, ended at 10:20 BST.
The row over the artificial reef has led to tensions between the UK, Gibraltar and Spain in recent weeks.
Spanish fishing boats set off at about 09:00 BST to protest near the spot where 70 concrete blocks were dropped into the sea to create the artificial reef.
It had been suggested that they might try to remove the blocks, but the fishermen said before the protest that they would not attempt this.
The Spanish government has accused Gibraltar of creating the reef, which is located off the western runway of Gibraltar Airport, "without the necessary authorisation" in "waters that are not theirs".
It has said that, in building the reef, Gibraltar is contravening environmental laws and damaging Spain's fishing industry, because Spanish fishing nets are in danger of catching on the concrete reef blocks.
But Britain has said it was trying to encourage sea-life to flourish.
In recent years the dispute has taken the form of a clash over fishing rights. Both Spain and Gibraltar have claimed jurisdiction over the waters off the Rock, and both sides have complained about incursions into what they claim are their waters.