Eastwood's own speech at last week's Republican National Convention drew mixed reviews for a section where he debated an empty chair standing in for Obama.
During one section of the speech, Eastwood asked the empty chair: "What? What do you want me to tell Romney? I can't tell him to do that to himself. You're absolutely crazy!"
Obama later responded by insisting he was not offended by Eastwood's attack and remained a fan of the Gran Torino director.
Harold & Kumar star Penn – who briefly worked in Obama's Office of Public Engagement – took aim at Eastwood while defending the president's record at the DNC.
"I've worked on a lot of fun movies but my favourite job was having a boss who gave the order to take out Bin Laden and is cool with all of us getting gay married," Penn said of Obama.
He then joked: "So thank you invisible man in the chair for that!"
The Daily Show's Jon Stewart also mocked Eastwood last week, claiming that the filmmaker's speech was the "most joy [he's] gotten from an old man since Dick Cheney non-fatally shot one in the face".
Earlier this week, the Obama campaign released a new video which sees the president meeting Harold and Kumar (John Cho, Kumar).
Watch Kal Penn's DNC speech below: