Two workers were giving out polio drops in a suburb of Peshawar when gunmen on a motorbike rode up and sprayed them with bullets, causing one serious injuries, Jan Baaz Afridi, doctor in charge of the immunisation campaign in Khyber Pakhtunkhwa province, told AFP.
Senior police official Asif Iqbal confirmed the attack and said police were investigating the motive and who was behind it.
Two other polio teams were targeted in similar attacks in the towns of Nowshera and Charsadda, police said, but the health workers escaped unharmed. One passerby was slightly injured in Nowshera.
Pakistan, one of only three countries in the world where polio remains endemic, launched a three-day nationwide immunisation campaign, supported by the UN, to tackle the crippling disease on Monday.
But violence has blighted every day of the campaign so far: one health worker was shot dead in Karachi on Monday and four more were killed in the city with another gunned down in Peshawar on Tuesday.
Efforts to tackle the highly infectious disease in Pakistan have been hampered over the years by local suspicion about vaccination.
The Taliban banned immunisations in the tribal region of Waziristan, condemning the campaign as a cover for espionage after the jailing of a doctor who helped the CIA find Osama bin Laden using a hepatitis vaccination programme.