"If they do that, if I think it's at all a hostile act, I will put very serious tariffs or stop trading with Europe on many things," Trump told reporters on the way to an event in North Carolina, calling the proposal "laughable".
"If it's a good intention, that's fine. If it's a bad intention, we'll put very heavy tariffs on Europe," he said.
Von der Leyen made the proposal on Wednesday during her annual State of the Union speech, attended by Canadian Prime Minister Mark Carney, as she sought to deepen ties among allies in what she called "an openly hostile world".
The EU faces the risk of war on its borders due to Russia's invasion of Ukraine, while both the bloc and Canada have grappled with trade tensions with the United States and an increasingly assertive China.
Earlier in September, Carney had said he was seeking a "unique alliance" with the EU as relations between Ottawa and Washington have deteriorated since Trump's return to the White House, leading to tariffs on tens of billions of dollars in cross-border trade.
The EU has historically resisted flexible alliances that have no legal status.
One EU diplomat expressed surprise at the proposal saying "nobody really knows what it means", adding there were concerns that the European Commission chief "is over-promising and won't be able to deliver".
Carney is due to address the European Parliament on Thursday morning.
