Trenches ran directly through the front part of the park, and the building of the former animal feed factory on the grounds was requisitioned. Originally used as a warehouse, it was now repurposed to prepare meals for the thousands of soldiers stationed around the park. Additionally, military horses were stabled there.

In the eight years since its opening, the park had grown into a full-fledged zoo, home to all the major animal species, preferably in large numbers. However, the presence of so many predators was considered a life-threatening risk by the Dutch military leadership. In the event of a German attack, the animal enclosures would likely be hit by shelling, and the risk of the animals escaping was too great. Therefore, they had to be preemptively euthanized.

With a heavy heart, director Cornelis Ouwehand himself carried out the order. He preferred to euthanize the animals himself rather than have the military do it. The only exceptions were the polar bears—a mother with her two cubs—whom he managed to save. In his book Fatherland Speaks to the Youth, published after the war, Cor Ouwehand wrote:

"Amid the roar of German planes still flying overhead, accompanied by the thunderous anti-aircraft fire, I make the heavy journey past the predator enclosures, controlling my nerves as best I can to avoid causing the animals unnecessary suffering. One by one, my lions, tigers, and bears fall..."

Major Landzaat and the Pavilion

On May 10, 1940, Ouwehand and his staff were ordered to evacuate the park. The bunker beneath the residential house was requisitioned by the local military commander. The intense battles in the days that followed resulted in most of the zoo being destroyed, despite the brave efforts of Major Willem Pieter Landzaat (1892–1940), commander of the 1st Battalion of the 8th Infantry Regiment. During the Battle of the Grebbeberg, he and his men defended the last defensive point: the so-called "pavilion" at the entrance of Ouwehands Dierenpark. Under heavy fire, he held his ground, even as the situation became hopeless. Ultimately, he was fatally struck while continuing to issue orders until the very end.

After the surrender, Ouwehand began rebuilding his park. It reopened in 1942, but when the Allied forces approached in 1944, German soldiers wanted to shoot the newly acquired predators and slaughter the other animals for food. To save as many animals as possible, the keepers moved them in a caravan from Rhenen to Veenendaal. By the end of the war, a significant number of animals had survived the battles, allowing Ouwehand to rebuild his park for the third time.

The widow of Major Willem Pieter Landzaat later had a small white house built on the site in his memory. This house still stands today. Major Landzaat was posthumously awarded for his bravery and self-sacrifice. He is buried at the Military Cemetery of Honor Grebbeberg.