The Hampstead Football Club was founded in 1866 and the first recorded game took place in 1867. The club was re-named Harlequin Rugby Football Club four years later, allegedgly because the membership was no longer purely local but the HFC monogram had to be retained. The word 'Harlequin' was found in a dictionary and all present agreed to the new name. This caused a split in the membership and the half that did not form the Harlequins went off and formed a club known as the Wasps.

In 1906, the club was invited by the Rugby Football Union to use the new national stadium in Twickenham. In the early days only one or two internationals played there during the season, and not long after Twickenham became the headquarters of the Harlequin Football Club.

In 1963, Harlequin acquired an athletics ground over the road from the Twickenham ground, which became its training pitch. This subsequently become their home: the Stoop Memorial Ground. The stadium was named after Adrian Dura Stoop, who won 15 caps for England and is said to have been the person who developed modern back play.

On the field, and with the introduction of leagues in 1987 bringing with it a more competitive environment, the Quins had maintained their status in the Premier Division as one of England's top 12 clubs until 2005.

The club has won the Rugby Football Union clubs knockout competition on two occasions: the John Player Cup in 1988 and Pilkington Cup in 1991. In addition, they played in the finals of 1992, 1993 and 2001.

The Quins became the first British team to win the European Shield in 2001, defeating Narbonne 42-33 in the final. They then became the first team to win the tournament twice, defeating Montferrand 27-26 in the final of the renamed Parker Pen Challenge Cup on 22 May 2004.

It was in 2005-06 season, the Quins were relegated to National Division One after finishing at the bottom of the Zurich Premiership. In July of that year they announced that they would be establishing a partnership with rugby league club London Broncos, which saw the two clubs sharing Harlequins home ground of The Stoop from the start of the 2006 Super League season. As part of the deal, the Broncos changed their name to Harlequins Rugby League, though the two clubs remain under separate ownership.

In 2005-06, Quins dominated National Division One. They won 25 of their 26 league matches, including their first 19, losing only at Exeter Chiefs on 25 February 2006. Quins also averaged nearly 40 points per match, scored four or more tries in 20 matches, and racked up an average victory margin of slightly over 25 points. They secured their return to the Premiership on 1 April with four matches to spare.

In the 2007-08 season Harlequins won 12 of their 22 Guinness Premiership matches and finished 6th in the league. Harlequins got off to a shaky start which saw them in 2nd, 3rd 4th place consecutively, and during the second half of the season Harlequins managed to reach 3rd after a string of 7 out of 9 wins, but defeats from London Irish, Sale Sharks and Leicester Tigers to finish the season meant that Quins dropped to 6th and missed out on the play offs.

Quins finished second in the 2008-09 Guinness Premiership table. In the play-offs, they lost 0-16 at home to eventual losing finalists London Irish.

Quins also hosted their first "Big Game" at Twickenham over the Christmas period, playing out a 28-28 draw with Leicester Tigers in front of 52000 people.

In the 2008-09 Heineken Cup Harlequins came top of their pool, including beating tournament favourites Stade Français both at home and away in front of 80000 people in the Stade de France in Paris. They lost 5-6 at the Stoop to eventual tournament winners Leinster Rugby at the quarter final stage, In which the infamous Bloodgate Scandal took place.

The contrast between the 2009-10 season and the previous season could hardly be greater. With the shadow of Bloodgate still hanging over the club, the Quins struggled to an 8th place finish despite retaining most of the players from their successful previous campaign. They also made a swift exit from the Heineken Cup at the group stages whilst failing to chalk up a single victory in the competition. Owing to the club's lower league position, they failed to qualify for the competition for the first time in three years.

Quins also hosted their second "Big Game" at Twickenham. Despite losing 20-21 to "London" Wasps, the game attracted 76000 spectators.

Following the resignation of Dean Richards in August 2009, Conor O'Shea was appointed Director of Rugby in March 2010