Italian authorities have ordered the seizure of the migrant rescue ship Mare Jonio after 49 migrants were brought to safety in an Italian harbour with the vessel.
The ship has been escorted to the port of Lampadusa where the migrants are expected to disembark.
The first interrogations of the ship's crew could take place on Tuesday night, according to Interior Ministry sources.
State prosecutors are investigating a possible case of abetting illegal migration, according to the ANSA news agency.
"In Italy there is now a government that understands borders and is making sure that laws are observed, particularly by human traffickers," Interior Minister Matteo Salvini said on Tuesday. "He who makes mistakes, pays."
The non-governmental organisation Mediterranea picked up the migrants on Monday from a rubber boat in distress some 49 nautical miles off the Libyan coast.
The migrants "can be cured, fed, clothed, given all kinds of comfort goods but, as far as I'm concerned, with my permission, they won't set foot in Italy," Salvini told SkyTG24 news channel.
However, one migrant with suspected pneumonia was allowed to leave the boat earlier in the day and is being treated by a doctor in Lampedusa, Federica Mameli, a Mediterranean spokeswoman, told dpa.
Salvini accused the NGO of "committing the crime of abetting illegal migration" by pre-empting a rescue operation by the Libyan coastguard, which would have seen the migrants returned to Libya.
It disobeyed Italian border police orders not to enter Italian waters, and instead of sailing towards Lampedusa, it should have headed to nearer Tunisian or Libyan shores, Salvini argued.
He threatened legal consequences: "If a citizen forces a police roadblock, they get arrested. I expect this to happen [for Mediterranea's crew]."
Since he took office in June, Salvini has repeatedly turned away NGO rescue ships from Italian ports, arguing that his country could not put up with any more migrants from North Africa.
On previous occasions - most recently in late January - NGO ships were eventually allowed in after other European Union nations agreed to take in some of their migrant passengers.
In Brussels, a European Commission spokeswoman repeated calls for EU governments to set up "predictable arrangements" to deal with these crises, rather than rely on ad-hoc agreements.
Australian Associated Press