The tankers were left vulnerable on the side of the road after Pakistan shut down a key border crossing.
About a dozen militants peppered the vehicles with automatic gunfire at a truck stop on the outskirts of the capital Islamabad. Some 20 trucks went up in flames and four people were killed and seven injured, authorities said.
Hours later, gunmen attacked and burned two other trucks carrying Nato supplies in south-west Pakistan, killing the driver.
There have been four such attacks since Pakistan closed its main border crossing into Afghanistan to Nato supply convoys last Thursday.
The closure was an apparent reaction to a series of alleged Nato incursions, including a helicopter attack that killed three Pakistani soldiers. Traffic has since been backing up at various points along the route from the southern port city of Karachi to the crossing at Torkham - where scores of trucks remain stranded and vulnerable to attack in the volatile Khyber Pass.
Although Pakistan says the Torkham blockade will soon be lifted, the latest attack and the Taliban threat seemed certain to raise the stakes in the closure, which has exacerbated tensions between Washington and Islamabad.
Convoys crossing from Pakistan bring fuel, military vehicles, spare parts, clothing and other non-lethal supplies for foreign troops.
"We are trying our best to protect the places where vehicles have accumulated, and we are not dispatching any more trucks from Karachi for now," said Shakir Khan Afridi, president of the Khyber Transport Association, a major umbrella organisation representing some 7,000 truckers.
Islamabad lies about 120 miles from Torkham.
The Pakistani Taliban, which last week threatened more attacks on the supply lines, claimed responsibility for the Islamabad attack in a telephone call to an Associated Press reporter.
Spokesman Azam Tariq said a new wing of the group had been created to strike the convoys and that the attacks "would continue until the supplies are completely stopped".
Trucks moving supplies from Karachi through Pakistan into Afghanistan make frequent stops along the way for their drivers to rest along the several-day journey, and Kalim Imam, police chief of the capital Islamabad, said it was impossible for police or local authorities to protect them all the time.
"This entire thing is very vulnerable for such attacks," he said.
There was no immediate claim of responsibility for today's second attack on the two trucks heading to the Chaman border crossing in the south-west, which has remained open.
Mohammad Hashim, a government official in the in the south-west district of Kalat where the attack took place - about 100 miles south of the region's main city of Quetta - said two gunmen on a motorcycle opened fire on the trucks, then torched the vehicles, killing one driver. The unidentified gunmen fled.
One of the trucks was carrying water while it was not yet clear what the other truck's cargo was, he said.
While Nato and the US have alternative supply routes into landlocked Afghanistan, the Pakistani ones are the cheapest and most convenient. Most of the coalition's non-lethal supplies are transported over Pakistani soil after being unloaded at docks in Karachi. There are some 140,000 international forces currently in Afghanistan.
In addition to the Torkham and Chaman crossings from Pakistan, Nato also receives supplies via the Central Asian states north of Afghanistan.
Afridi said, however, that some trucks on their way to Chaman have also been unable to get through due to the massive flooding in the region, which left millions homeless and destroyed thousands of miles of roads.
"On that route too, container trucks and oil tankers are stopped at different points but not in very large number," he said.
On Friday, a day after the closure of the Khyber Pass route to Nato and US traffic, there were two attacks on oil tankers headed to the country, one of which was claimed by the Pakistani Taliban.
The Pakistani Taliban are the country's largest militant group. Based in the north-west, it has claimed responsibility for scores of suicide bombings against Pakistani government and security targets, as well as Western ones. The group has ties with the Taliban movement in Afghanistan that is fighting the US-backed government there.
Striking the supply line now gains the group more media attention than normal and makes the mission in Afghanistan appear vulnerable.
While attacks on convoys in Pakistan give militants a propaganda victory, coalition officials say they do not result in shortages in Afghanistan. Hundreds of trucks still cross into Afghanistan each day.
Some attacks are believed to be the work of criminals, who can sell much of the vehicles, clothes and other goods they carry. Officials have alleged truck owners may be behind some of the incidents, perhaps to claim insurance fraudulently.
Yesterday, Pakistan's ambassador to the US, Husain Haqqani, said the border crossing would soon be reopened.
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