Kyiv said that a Russian missile attack had killed Spanish national Emma Igual, and Anthony Ihnat, a Canadian citizen, both working for the NGO Road to Relief.
Two other volunteers with the group were said to have been injured during the incident in the eastern Donetsk region.
The NGO, which helps evacuate wounded people from front-line areas, said its four volunteers were trapped inside the van as it flipped over and caught fire after being struck by Russian fire near the town of Chasiv Yar.
A statement on its Instagram page said that Mr Ihnat died in the attack, while two others, German medical volunteer Ruben Mawick and Swedish volunteer Johan Mathias Thyr, were seriously injured.
Spain’s acting foreign minister Jose Manuel Albares told Spanish media that officials had received “verbal confirmation” of Ms Igual’s death.
Speaking in his nightly address, Mr Zelensky paid tribute to the deceased two aid workers.
“This Russian shelling once again confirms how close the war against Ukraine is for everyone in the world who truly values human life and who considers it a common moral duty of humanity to stop terror and defeat evil," he said.
The volunteers had been on their way towards Bakhmut, the eastern town which has seen some of the war’s bloodiest fighting, when they were struck, said Road to Relief.
It came as Russia fired nearly three dozen drones at Kyiv early on Sunday.
Ukraine’s air force said the military destroyed 26 out of 33 drones launched by Russia, all of which targeted Kyiv and the region around it.
Fragments from downed drones fell on five different districts, said Mayor Vitali Klitschko and the city’s military administration, with one person injured.
Kyiv region Governor Ruslan Kravchenko wrote on Facebook that an unspecified infrastructure facility and eight houses had been damaged as a result of the overnight strikes.
There was no immediate comment from Russia about the attacks.
In February, US national and medic Pete Reed was killed near Bakhmut when his evacuation vehicle was hit by a missile.
In May, AFP video journalist Arman Soldin was also killed in Chasiv Yar near Bakhmut by missile fire.