"I'm not a fan of hers... and she probably has heard that," Trump said of Markle, before adding, "I wish a lot of luck to Harry 'cause he's gonna need it."
Trump previously spoke about Markle last year, calling her "nasty" after she blasted him as "divisive" and "misogynistic."
"She was nasty to me, and that's OK for her to be nasty,. It's not good for me to be nasty to her and I wasn't," he told Good Morning Britain's Piers Morgan. "You know what? She's doing a good job, I hope she enjoys her life... I think she's very nice."
At the time, Trump praised Prince Harry as a "terrific guy."
"He spent a lot of time talking to [my daughter] Ivanka and talking to my family," Trump said. "He couldn't have been nicer... I think he's great."
Trump's most recent comments come after the couple made a video about the importance of voting ahead of the November election, in which the current president will face off against Joe Biden.
"We're just six weeks out from Election Day, and today is National Voter Registration Day. Every four years, we are told the same thing, that 'This is the most important election of our lifetime,'" Markle said. "But this one is. When we vote, our values are put into action and our voices are heard. Your voice is a reminder that you matter. Because you do. And you deserve to be heard."
"As we approach this November, it's vital that we reject hate speech, misinformation and online negativity," the duke said, with his wife adding, "So as we work to re-imagine the world around us, let's challenge ourselves to build communities of compassion."