"If NGOs do not sign up (to a new code of conduct), it is difficult to see how they can continue operating," Interior Minister Marco Minniti said in an interview with Turin daily La Stampa.
Minniti's warning came a day after Italian authorities impounded a boat operated by German aid organisation Jugend Rettet on suspicion its crew effectively collaborated with people traffickers in a way that facilitated illegal immigration.
The aid organisation, which has only been operational for a year, said it would seek to overturn the seizure.
"Our Italian lawyer is appealing the confiscation of our boat. Our first priority is to free it and resume our rescue missions," a spokeswoman told AFP.
Italian authorities had been monitoring Jugend Rettet's boat, the Iuventa, since October.
Its crew is suspected to taking on board dinghy loads of migrants delivered directly to them by people traffickers and allowing the smugglers to make off with the vessels to be used again.
At least one such meeting allegedly took place only 1.3 miles off the Libyan coast, according to the prosecutor's file, the contents of which were published by Italian media.
The crew are also alleged to have flouted the authority of the Italian coastguard, which oversees rescue operations in the zone, out of humanitarian zeal rather than for any other motives.
Under the code of conduct, boats like the Iuventa would notably have to have an Italian police officer on board monitoring their activities.
Four of the nine NGOs involved in search and rescue operations have so far agreed to abide by the code: Save The Children, Malta-based MOAS, Spain's Pro-Activa Open Arms and Germany's Sea-Eye.
Pro-Activa said Thursday it regarded the new rules as unnecessary but acceptable as they would not involve any "significant change or impediment" to its rescue operations.
Among those who have refused to sign is the Nobel Prize-winning organisation Doctors Without Borders (MSF).
'Light at the end of the tunnel'
Minniti meanwhile said a sharp fall over recent weeks in the number of migrants arriving in Italy following rescues was an indication that efforts to beef up the Libyan coastguard and cooperation with local mayors was bearing fruit.
"In recent days we have begun to see light at the end of the tunnel," the minister said.
He revealed plans for further talks this month with Libyan mayors on economic development initiatives and with Chad, Niger and Mali on measures to reduce the number of migrants leaving those countries in the hope of reaching Europe.
Italy sent a naval patrol boat to the Libyan capital Tripoli on Wednesday to begin discussing further support for the Libyan coastguard's efforts to stop traffickers.
The move, the first in a larger naval support mission, prompted an angry response from Khalifa Haftar, the military strongman who controls parts of eastern Libya.
A spokesman said Haftar had issued an order to his forces to prevent foreign vessels entering Libyan waters without his permission.
Italian officials played down the significance of the rhetoric. The mission has been agreed with the UN-backed Libyan government based in Tripoli, whose authority is contested by Haftar.
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