Reading from a pamphlet, a Taliban spokesman Abd al-Samad on Thursday said Karzai and those working for him were infidels and the puppets of the United States.
"They are the agents of the US and its allies," he said.
"For the last time we inform you to advise your family members and relatives to stop working for them, they will be forgiven. Otherwise be ready for any attack and harm," Abd al-Samad said.
The Taliban warned they would attack the United Nations, the US-led forces, aid workers, non-governmental organisations and the Afghan national police and army.
The pamphlet said those people who previously worked for the country's Communist government were now working for Karzai.
The pamphlet was issued by a faction of Taliban which calls itself the southeastern zone Mujahidin.
The latest warming comes amid a deteriorating security situation sweeping Afghanistan.
The war-ravaged country's southern and southeastern parts have particularly witnessed a spurt in attacks and kidnappings.
A bomb blast in Kandahar on 6 January killed 15 people and injured many.
The renewed violence has fuelled speculations the Taliban –ousted from Kabul in December 2001 by US-led forces- and remnants of the outlawed al-Qaida were regrouping in Afghanistan's mountainous countryside.
The wave of violence has forced UN and humanitarian agencies to scale down their operations in many regions.
Even the capital Kabul has not been spared of the escalating attacks.