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 Tuesday night, according to Interior Ministry sources.
State prosecutors are investigating a possible case of abetting illigal 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 Tuesday. "He who makes mistakes, pays."
The non-governmental organization (NGO) Mediterranea picked up the migrants on Monday from a rubber boat in distress some 49 nautical miles (78 kilometres) 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 earlier Tuesday.
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 preempting 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.
"The commission renews once again its call on all member states to bear first and foremost the humanitarian imperative in mind and contribute to a swift resolution of the situation," she said.
Salvini claims that his closed-door immigration policy has contributed to a dramatic drop in migrant arrivals and in migrant deaths at sea.
On Monday, his office said there were 335 migrant landings between January 1 and March 15, a nearly 95-per-cent reduction from the same period of 2018.
But preventing migrants from leaving Libya or returning them there is controversial because it puts them at risk of suffering serious human rights violations, including slave labour, beatings and torture.
Hampering NGO rescue work in the Mediterranean is also making migrants' journeys more risky, according to human rights organizations.
Some 1,280 people left from North Africa to Europe in the year to date, and at least 154 died at sea, Flavio Di Giacomo, a spokesman for the International Organziation for Migration, tweeted.
Noting that "another shipwreck happened today off the Libyan coast," in which several were feared dead, Di Giacomo said: "The death rate is impressive."