All were born in the US except for Ms Omar, who arrived in the US as a child after fleeing Somalia with her family.
Mr Trump described each Congresswoman as a "force of evil", addressing them each individually.
"These left-wing ideologues see our nation as a force of evil. The way they speak so badly of our country. They want to demolish our Constitution. Weaken our military. Eliminate the values that built this magnificent country. You have to look at some of their recent comments, which are never talked about. When you see the four congresswoman, oh isn't that lovely," he said.
He chided Ms Tlaib for calling him a "motherf**ker", saying "that's not somebody that loves our country". He criticised Ms Ocasio-Cortez for referring to squalid immigration detention centre facilities on the US-Mexico border as "concentration camps". He also accused Ms Omar of being soft on terrorism.
"Omar laughed that Americans speak of al-Qaeda in a menacing tone," he said. "You don't say America with this intensity. You say al-Qaeda makes you proud. Al-Qaeda makes you proud. You don't speak that way about America," he added, referring to her remarks in a 2013 interview.
As he spoke about Ms Omar, he drew a chant from the crowd of, "Send her back! Send her back!"
Ms Omar has since responded to the jab on Twitter, with a quote from civil rights activist Maya Angelou: "You may shoot me with your words, you may cut me with your eyes, you may kill me with your hatefulness, but still, like air, I'll rise."
Former director of speechwriting for President Barack Obama and host of Pod Save America podcast Jon Favreau described the atmosphere at the rally as "chilling".
"The crowd at Trump's rally chanting 'send her back' after the President viciously and dishonestly attacked Ilhan Omar is one of the most chilling and horrifying things I've ever seen in politics," he said.
Mr Trump also told the rally he had "lost all" of his friends after winning the presidency.
"I lost all my friends. They've tightened up. They've choked. They can't breathe," he said. "Because people have such respect for the office of the presidency. They have respect for office of the president. They have respect for our country again, our country is respected again."
Over the weekend, Mr Trump sparked a firestorm after he posted a series of racist tweets implying the four women were not natural-born American citizens.
In a series of tweets, the US President suggested the new generation of Democrats who have been feuding with House Speaker Nancy Pelosi should "go back" to their countries to fix their governments, saying "you can't leave fast enough".
"So interesting to see 'Progressive' Democrat Congresswomen, who originally came from countries whose governments are a complete and total catastrophe, the worst, most corrupt and inept anywhere in the world (if they even have a functioning government at all), now loudly and viciously telling the people of the United States, the greatest and most powerful Nation on earth, how our government is to be run," he wrote.
"Why don't they go back and help fix the totally broken and crime infested places from which they came. Then come back and show us how it is done.
"These places need your help badly, you can't leave fast enough. I'm sure that Nancy Pelosi would be very happy to quickly work out free travel arrangements!"
