Results reveal that 61.1 percent of those that voted were against an Association Agreement with Ukraine, and 38.1 percent were in favour.
The agreement grants non-EU members privileges in economic, political and cultural spheres, and is considered a step towards EU membership.
Krzysztof Szczerski, foreign policy advisor to President Duda said the result “is very bad news, because it shows that what was a key goal for Poland, namely bringing Eastern Partnership countries into the EU, is meeting with resistance in countries at the heart of Europe.
“It’s bad news for Europe and bad news for Poland,” he added.
All of the other 27 EU states besides the Netherlands have already ratified the agreement.
Although only 32 percent of voters turned out in the Netherlands, this is above the 30 percent threshold needed for the result to be binding.
The result has been interpreted as a sign of growing euroscepticism, and it is problematic for the Dutch government, as parliament had already approved the Association Agreement.
The referendum was permitted after 400,000 signatures were gathered in an action coordinated by Eurosceptic activists.