Forces loyal to Yemen's president opened fire on protesters in the southern city of Taiz over the weekend, bringing the death toll since Sunday to at least 25.
Amateur video showed masked men firing rifles from rooftops. At least four protestors were killed in Monday's attack, according to the Associated Press.
The violence flared up after President Ali Abdullah Saleh again refused to sign an agreement to hand over power.
Saleh's spokesman Ahmad Al Sufi claimed the deaths were the result of "Muslim Brotherhood attempting to free criminals from prisons."
The number of deaths from the attacks have varied, but late Tuesday United Nations human rights chief Navi Pillay said the death toll could be more than 50.
Pillay said the killings occurred when government-affiliated forces destroyed a protest camp in Taiz.
"Reports indicate that hundreds more have been injured," he added. "Such reprehensible acts of violence and indiscriminate attacks on unarmed civilians by armed security officers must stop immediately."
President Saleh has clung to power despite almost four months of mass protests against him.
Saleh has repeatedly warned that Islamic militants and al Qaeda would seize control of the country if he steps down.