Mr Trump said "it would show great weakness" if Israel allowed entry to Ilhan Omar and Rashida Tlaib.
The move comes as a reversal, after the Israeli ambassador to the US said last month that they would be allowed in.
Israel bans supporters of the movement boycotting Israel, something the pair have backed.
The move was confirmed in a statement from the Israeli Interior Ministry, which said it was "inconceivable that those who wish to harm the state of Israel while visiting would be granted entry".
Last month Israeli Ambassador Ron Dermer said the two Democrats would be permitted to visit "out of respect for the US Congress and the great alliance between Israel and America".
President Trump, who has a close relationship with Israeli Prime Minister Benjamin Netanyahu, has frequently feuded with the lawmakers and in remarks widely condemned as racist, told them to "go back" to the countries that their families were from.
On Thursday, he took to Twitter to urge that they be blocked, adding that "they hate Israel & all Jewish people, & there is nothing that can be said or done to change their minds".
According to US media, their trip was meant to begin on Sunday, and would include a stop at one of the most sensitive sites in the region - Jerusalem's Temple Mount, which is known to Muslims as Haram al-Sharif.
They also planned to visit Israeli and Palestinian peace activists and travel to Jerusalem and the West Bank cities of Bethlehem, Ramallah and Hebron.
The trip to the West Bank was planned by Miftah, an organisation headed by Palestinian peace negotiator Hanan Ashrawi.
Ms Tlaib was planning to stay for two extra days to visit her grandmother, who lives in a Palestinian village.
On Wednesday, Mr Netanyhau held consultations with the country's foreign minister, interior minister, National Security Council chief and attorney general, according to Haaretz newspaper.
Israeli law blocks entrance visas to any foreigner who calls for any type of boycott that targets Israel - either economic, cultural or academic.
The law attempts to suppress the "boycott, divest, sanction" movement, which has drawn growing support across Europe and the US.
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