"'Open Arms' has been sailing the Mediterranean for six days and is now threatening to come to Italy.
"It has had all the time it needed to sail to Spain, its homeland, the country whose flag it flies and where several mayors have said they will take in the migrants," he went on.
"Evidently the lives of those on board are not their real priority," Salvini said, accusing Spanish NGO Proactiva Open Arms of "political provocation".
"Italy's waters are closed to the Open Arms and we are ready to impound the ship," he underlined.
Salvini's warning came after Malta on Monday refused to allow the 'Open Arm's to dock at a port on the island, prompting the NGO to tweet: "Better to receive a fine than to be complicit (in this situation)."
Under Italy's newly enacted security law, NGO vessels that enter Italian territory without permission will be confiscated and the charities operating the boats face fines of up to one million euros.
The United Nations refugee agency UNHCR on Tuesday voiced worry over the security law, which it warned risks deterring future lifesaving efforts in the Mediterranean.
The 'Open Arms' headed to Malta after Salvini late last Thursday signed an order banning the vessel from entering his country's waters.
Among those on the vessel are two babies and a further 30 minors.
Proactiva Open Arms founder Oscar Camps has called on European Union member states to take the migrants. He said he is concerned for their welfare and that they need "medical and psychiatric assistance".
( With inputs from IANS )