It is the second time in 10 months the zoo in Amersfoort has been forced to close because of escaping animals.
Guests were already inside the zoo on Monday when the wolves were spotted roaming around the park.
Eric Bax told Dutch broadcaster RTL Nieuws he saw one of them while he was with his girlfriend and young son.
“Suddenly we saw a wolf walking through the bushes - I grabbed my son and ran off,” he said.
“My girlfriend called the police straight away and together with some other visitors we put up a barricade.”
A spokesman for the zoo said the animals were “isolated, sedated and taken back to their enclosure”.
He added: “A wolf is not dangerous to visitors. They don’t attack people.
“We asked our visitors to go inside and ensure the situation was as calm as possible so that we could bring the situation to an end.”
In the previous incident, two chimpanzees were shot dead when they left their enclosure in November.