"Israel will be held fully responsible for any attack on Lebanon," the foreign ministry said in a letter to the United Nations.
Lebanon "cannot prohibit a ship from leaving its ports if its cargo, passengers and destination all comply with Lebanese law," it said in the letter.
Israel's ambassador to the United Nations warned the world body last week that the Jewish state was entitled to use "all necessary force" to stop the Lebanese activists' boats.
On Monday, Defense Minister Ehud Barak also reiterated a warning that Lebanon would be responsible for any "violent and dangerous confrontation" with vessels sailing to Gaza from its shores.
In defiance of the Israeli warnings, Lebanese civilian groups are still planning to transport aid, including medical supplies, by sea to the Israeli-blockaded Palestinian territory via Cyprus.
One of two boats, the "Naji al-Ali," on Monday received Lebanon's green light to depart for Cyprus on the first leg of the trip to Gaza, which is run by the Islamist movement Hamas. It is currently docked in the northern port of Tripoli.
The vessel would need Cypriot authorization to depart for Gaza from its shores, but officials in Cyprus said on Tuesday that the island was keeping in place a ban on ships departing for Gaza.
Organizers in Lebanon have said they may change course before reaching the island and head straight towards the Palestinian territory.
A second ship, the "Mariam", also plans to carry aid to Gaza in another attempt to break the four-year siege of Gaza with some 50 women activists on board, including 30 Lebanese.
The "Mariam" has not yet been given Lebanese clearance to sail.