An air strike by the Saudi-led coalition on the old quarter of the Yemeni capital killed five people on Friday and destroyed three houses in the UNESCO-listed heritage site.
UNESCO condemned the air strike on the historical area it described as "one of the world's oldest jewels" of Islamic culture, urging warring parties to respect Yemen's heritage.
Residents said the pre-dawn strike was the first direct hit on old Sanaa since the launch of the bombing campaign against Huthi rebels in late March.
The missile hit the Qassimi neighbourhood, which boasts thousands of houses built before the 11th century, an AFP journalist reported.
It did not explode but it still destroyed three three-storey houses and killed five residents, including a woman and a child, medics and witnesses said.
The target of the raid was not immediately clear amid conflicting statements from residents about whether rebels had occupied one of the houses hit.
"I am profoundly distressed by the loss of human lives as well as by the damage inflicted on one of the world's oldest jewels of Islamic urban landscape," said UNESCO director general Irina Bokova.
"This destruction will only exacerbate the humanitarian situation and I reiterate my call to all parties to respect and protect cultural heritage in Yemen," she said.
Bokova said she was "shocked by the images of these magnificent many-storeyed tower-houses and serene gardens reduced to rubble".
"The historic value and memories enshrined in these sites have been irreparably damaged or destroyed."
The old city has already suffered some damage from air strikes on nearby targets, including the defence ministry, prompting a protest from UNESCO in May.
Sanaa's old city, situated in a mountain valley, has been inhabited for more than 2,500 years and was a major centre for the propagation of Islam, boasting over 100 mosques, 14 public baths and more than 6,000 houses built before the 11th century.
It was inscribed on UNESCO's World Heritage List in 1986.
The multi-storey houses rising above stone ground floors were built of rammed earth and burnt brick. Each building is decorated with geometric patterns of fired bricks and white gypsum, inspired by traditional Islamic art.
Earlier this month, UNESCO condemned May 31 air strikes that hit the ancient Great Dam of Marib, which was first built in the 8th century BC, in the city that was once the capital of the kingdom of Saba.
The UN body said the attack on the dam came a week after the national museum in Dhamar, in central Yemen, was "completely destroyed".