The commander of Libya's self-styled national army has ordered his naval forces to confront any vessel entering the country's waters without permission.
An Italian naval ship, Commandante Borsini, entered Libyan waters on Wednesday.
Italy initially hoped to send six ships into Libyan waters but had to downsize the mission after objections from the administration in Tripoli.
Haftar, who controls most of eastern Libya, said the order was applicable to bases in Benghazi, Ras Lanuf and Tobruk as well as the capital Tripoli, in the west, which he does not control.
Italy sent the mission to help Libya's coastguard curb the flow of refugees and asylum seekers after a request by the UN-backed Government of National Accord (GNA) in Tripoli.
Paolo Gentiloni, Italy's premier, said Italy's deployment responded to a request by the GNA, which later denied striking a deal with the Italians.
Libya's eastern-based parliament, rivals to Tripoli and allied with Haftar, strongly opposed the move.
Returning refugees and asylum seekers back to Libya amounted to "exporting the illegal migration crisis to Libya", it said.
The development signals unresolved tensions between Haftar and Fayez Serraj, the GNA prime minister, who pledged to cooperate at a meeting outside Paris last week.
At the meeting hosted by French President Emmanuel Macron last week, Haftar and Serraj committed themselves to a ceasefire, to work toward presidential and parliamentary elections, and to establish a roadmap to secure Libya against "terrorism" and "trafficking of all kinds".
Al Jazeera's Mahmoud Abdelwahed, reporting from Tripoli, said Haftar's ability to translate his threats into action is open to question.
"Haftar does not have a military presence in the west and especially in the Libyan capital, Tripoli," he said, adding that forces loyal to Haftar near Tripoli would meet with resistance if they made any attempt to reach GNA-controlled ports.
He said the EU and the US are helping Tripoli-controlled government forces monitor Libyan waters and airspace.
"Haftar doesn't have navy forces in the west. The only ships he controls are in the east, off the Mediterranean oil ports and Benghazi," Al Jazeera's Abdelwahed said.
"With the Sophia delegation monitoring Libyan waters, Haftar cannot move his ships."
Operation Sophia is an EU mission to stop people trafficking and arms smuggling in Libya.
Officials hope boats being sent back to Libyan ports will have a powerful deterrent effect on would-be refugees and asylum seekers considering paying traffickers for passage to Europe.
Squalor and abuse
The approach has been criticised by international rights groups who say people returned to troubled Libya face detention in squalid camps and abuse at the hands of traffickers.
"After years of saving lives at sea, Italy is preparing to help Libyan forces who are known to detain people in conditions that expose them to a real risk of torture, sexual violence, and forced labour," said Human Rights Watch's Judith Sunderland.
Hundreds of thousands of refugees and asylum seekers are brought to Italy each year from Libya in overcrowded boats that are often not seaworthy.
The Switzerland-based International Organization for Migration says 94,802 migrants have reached Italy this year.
At least 2,221 people have drowned this year while attempting to cross the Mediterranean Sea from Libya.
Libya descended into chaos following the 2011 civil war that toppled and killed Muammar Gaddafi and the country is now split between rival governments and militias.