The rally is one of more than 600 being held worldwide on the president's first full day in office.
The aim is to highlight women's rights, which protesters believe to be under threat from the new administration.
Mr Trump attended a multi-faith service at Washington National Cathedral and then visited the CIA's HQ.
In a speech there he said he was "1,000%" behind its employees and also railed against a "dishonest" media. He did not refer to Saturday's protests.
Protesters swarmed the streets and metro stations of the US capital on Saturday as they headed to the National Mall to hear speeches from Scarlett Johansson, Ugly Betty star America Ferrera, Ashley Judd, Gloria Steinem and Michael Moore among others.
Latest: Mass protests in US
Organisers had originally sought a permit for 200,000 people but later said they were expecting as many as half a million, which would dwarf Friday's inauguration crowd, the Washington Post reported.
A planned march to the White House proved impossible as the entire route was filled with demonstrators.  Interim DC Police Chief Peter Newsham told Associated Press: "The crowd stretches so far that there's no room left to march."
But there are other protests in some 300 cities across the US, from New York to Seattle. The scale of the turnout in Chicago prompted police to designate it a rally rather than a march.
Organisers of a London rally said between 80,000 and 100,000 people had taken part there.
During his speech in Washington, Michael Moore ripped up a copy of the Washington Post, saying: "The headline was 'Trump takes power'. I don't think so. Here's the power. Here's the majority of America right here. We are the majority."
The singer Madonna also made an appearance, swearing several times in a speech carried live by major US TV networks.
"Yes I am outraged. Yes, I have thought an awful lot about blowing up the White House," she said.
America Ferrera told the crowd: "We march today for the moral core of this nation, against which our new president is waging a war."
Many women have knitted pink "pussy hats" - a reference to a recording that emerged during the election campaign in which Mr Trump talked about groping women.
Defeated presidential candidate Hillary Clinton sent a tweet of support.
Organisers of the Women's March on Washington said in a statement: "The women's march on Washington will send a bold message to our new government on their first day in office, and to the world that women's rights are human rights."
They said the protest would not simply be about women's rights and would attract "people of all genders, ages, races, cultures, political affiliations and backgrounds", with support from the likes of Amnesty International.
The rally is peaceful, easing fears of a repeat of the violence that occurred after the inauguration ceremony on Friday, when protesters smashed windows. More than 200 were arrested and six officers hurt.
Anti-Trump marches have already taken place in Australia, New Zealand and in Asian cities such as Bangkok.
Several thousand women and men joined a rally in central Sydney, with a similar number in Melbourne.
Women's March Sydney co-founder Mindy Freiband told the crowd there: "Hatred, hate speech, bigotry, discrimination, prejudicial policies - these are not American problems, these are global problems."
American expatriate Bill Scholer, protesting in Tokyo, told Reuters news agency: "Everything we value could be gone. It's time to speak your mind and concerns and to do our best to salvage the values we cherish in America."
London, Belfast, Cardiff, Edinburgh, Leeds, Liverpool, Manchester and Bristol were among the UK cities holding protests, while Barcelona, Rome, Amsterdam, Geneva, Budapest, Prague and Berlin were among the European cities.
Mr Trump's Washington National Cathedral service was scheduled to be his sole official engagement on Saturday, although it was then announced he would visit the CIA HQ in Langley, Virginia, in the afternoon.
In a speech there, he told about 400 employees: "There is nobody who feels stronger about the intelligence community and the CIA than me."
During the election campaign, Mr Trump had sharply criticised the intelligence agencies over their stance on alleged Russian involvement.
Mr Trump also talked up his yet-to-be-confirmed nomination for CIA chief, Mike Pompeo.
"You will be getting a total gem," he told the employees.
Mr Trump has already ordered agencies to ease the "economic burden" of the health laws known as Obamacare. His team also quickly overhauled the White House website.
The website revamp replaces Barack Obama's policies with Mr Trump's new agenda.
The new administration lists only six issues on the website - energy, foreign policy, jobs and growth, military, law enforcement and trade deals.
Critics point out that it makes no mention of civil rights, LGBT rights, healthcare or climate change.
Hundreds of thousands have massed for a "Women's March on Washington", part of a global day of protests against new US President Donald Trump.