He ordered a full probe into the “mass killing”, which a local official said was punishment to revellers attending a party with music and mixed-sex dancing.
“This attack shows that there are irresponsible members among the Taliban,” Karzai said in a statement.
The Taliban denied they had taken part in the attack, which Karzai’s office said took place in Kajaki district in the southern province.
“The victims were killed for throwing a late-night dancing and music party when the Taliban attacked,” Nimatullah, governor for neighbouring Musa Qala district, told Reuters.
Men and women do not usually mingle in ultra-religious Afghanistan unless they are related, and parties involving both genders are rare and kept secret.
The killings, 75km north of the provincial capital Lashkar Gah, occurred at the beginning of a violent 24 hours for Nato and Afghan authorities in which 10 Afghan soldiers were killed in a mass insurgent attack, also in Helmand, while two US soldiers were slain by a rogue Afghan soldier.
Taliban spokesman Qari Yousuf denied the group was involved. “I spoke to our commanders in those villages, but they know nothing of the event.”
During their five-year reign, ended by US-backed Afghan forces sparking the present Nato-led war, the Taliban banned women from voting, most work and leaving their homes unless accompanied by their husband or a male relative.
Though those rights have been painstakingly regained, Afghanistan remains one of the worst places on Earth to be a woman.
Some democratic freedoms have also been wound back in what rights groups fear is an effort to reach a political reconciliation and possible power-sharing with the Taliban, who had also banned music and dancing.
Taliban gunmen stormed a lakeside hotel near Kabul in June demanding to know where the “prostitutes and pimps” were during a party, witnesses said. Some 20 people were killed.
In another setback for Nato, an Afghan soldier shot dead two US troops in east Afghanistan yesterday, the latest in a series of insider killings that have strained trust between the allies ahead of a 2014 handover to Afghan security forces.
The deaths in Laghman province brought to 12 the number of foreign soldiers killed this month, prompting Nato to increase security. There have been 33 attacks so far this year that have led to 42 coalition deaths. That is a sharp increase from 2011, when 35 coalition troops were killed, 24 of them American.