Two gunmen riding a motorcycle fired at the vaccinators at Badabher area on the outskirts of Peshawar while they were administering polio drops to children.
A female vaccinator was killed instantly, police and witnesses said.
The injured woman was taken to Lady Reading Hospital, where doctors described her condition as critical.
No group claimed responsibility for the attack.
Security forces cordoned off the area and launched a search operation for the gunmen.
On May 20, suspected militants attacked a team of polio vaccinators in the restive Bajaur tribal region and killed a security personnel.
Last month, a policeman was killed and another seriously injured when suspected militants attacked a team of polio vaccinators in Mardan town of Khyber-Pakhtunkhwa.
In January, six women and a man working for a NGO involved in the vaccination campaign were shot dead in Swabi district.
In December, eight volunteers involved in a polio vaccination drive were gunned down in a string of attacks in the southern port city of Karachi and Khyber-Pakhtunkhwa.
Last year, the Pakistani Taliban banned polio vaccinations in parts of Waziristan tribal region, claiming the drive was a cover for espionage.
Pakistan, Afghanistan and Nigeria are the only countries where polio is still endemic.