Thousands of people lined the streets and sang songs to remember the victims of the attack on Pulse, a gay nightclub in the Floridian city.
Soho is the historic heart of London’s gay scene and has also suffered attacks at the hands of homophobic extremists.
Mourners also gathered in major cities across the country, including Birmingham, Bristol and Manchester.
Old Compton Street fell silent at 7pm as a sign of respect for the partygoers killed on Sunday morning. Flags bearing messages of hope fluttered in the wind and a number of the fists were raised defiantly in the air during the two-minute silence.
After the people gathered on the packed road - home to a number of prominent gay clubs - finished observing the two minutes of respectful quiet, 49 balloons - one for each person killed - were released into the air.
A rendition of Simon and Garfunkel's hit Bridge Over Troubled Water was sung by London Gay Men's Chorus as the silence came to an end, followed by the road chanting: "We're here, we're queer, we will not live in fear."
Dozens of candles laid by mourners illuminated the St Anne's churchyard in the early dusk, while flowers were piled up at their side.
Labour leader Jeremy Corbyn, mayor of London Sadiq Khan and Education Secretary Nicky Morgan were among a cross-party group of politicians at the event.
Mr Corbyn said: "It’s an extraordinary turnout of people showing their solidarity against this awful crime and there is an amazing sense of coming together and unity here in London tonight, indeed as it is all over the world.
"We have to live in a society where homophobic hate crime is a thing of the past and the deaths that happened in Orlando are a sign of something deeply awful.
"We're here in Old Compton Street because of what happened here and it's that sense of solidarity that we've got.
"Love, in the end, defeats this crime, because it's stronger."
In 1999, a nail-bomb was detonated on the street, killing two people and injuring dozens more.
Following the two minutes of silence, the group - which also featured shadow chancellor John McDonnell and Scottish Conservative leader Ruth Davidson - laid flowers at St Anne's Church to those killed in the atrocity.
Ms Morgan, who is also equalities minister, said of her decision to appear alongside members of opposing parties: "I think this sort of tragic, horrific event in Orlando transcends normal party politics and we all wanted to be here to offer our solidarity with the people of Orlando who have suffered the most awful hate crime, fuelled by prejudice.
"I think it is very important - and quite moving - that the streets of London were filled with so many people who wanted to pay their tribute."
Press Association contributed to this report.
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