Donna, 42, was put down Tuesday, WXII-TV, Winston-Salem, reported. She had been diagnosed with cancer more than two years ago.
Katie, 36, was euthanized Wednesday, officials said. She was receiving radiation treatment for an inoperable tumor in her reproductive organs.
"She had stopped eating and would not respond to keepers," Dr. Mike Loomis, the zoo's chief veterinarian, said.
The zoo in Asheboro in central North Carolina lost a third female gorilla in May. Hope, 35, the only gorilla to give birth in the North Carolina Zoo, died of conditions related to her age.
"This is a sad day for all of us at the zoo and in many ways, represents the passing of an era with the loss of these members of our gorilla family," said David Jones, the zoo director. "These venerable ladies have been with us a long time.
The zoo is expecting to get two female gorillas next year from the San Diego and Oklahoma zoos. They will join North Carolina's male gorilla, Nikosi, 18, who is reportedly healthy.