University College London (UCL) Urban Laboratory found the number of LGBTQ+ venues fell from 127 to 53 since 2006 - a 58% decrease. This compares with a 25% drop in London pubs since 2001.
The report found said a "significant number" were closed due to property developments and increasing rents.
Sadiq Khan said the importance of LGBTQ+ venues "cannot be overstated".
The Mayor of London said LGBTQ+ venues "enrich London as a whole" and help "members of an often vulnerable community to take pride in their identity".
Ahead of the Pride parade on the weekend, Mr Khan and Night Tsar Amy LamÃ© announced a new LGBT+ Venues Charter, where owners and developers can commit to measures to protect their venues.
An annual audit to better track venue numbers was also announced by City Hall.
Last November, UCL highlighted a string of long-standing venues closing in rapid succession in the capital - including the Black Cap, the Queen's Head and Madame Jojo's.
21%
property development
9% changed terms of lease
6% business rate changes
5% licencing disputes
25% no information
Islington saw an 80% drop in LGBTQ+ venues since 2006, Lambeth venues fell by 47% while both Camden and Westminster lost 43% over the same time period.
As of 2016 there are no LGBTQ+ venues in 19 out of London's 32 boroughs - up from 10 in 2006.
Dr Ben Campkin, director of UCL Urban Laboratory, said: "A large number of venues have succumbed to changes of use, development, and the inability of operators and customers to defend themselves against rent increases and unfavourable changes to their terms of lease."
He added the report disputed claims that "LGBTQ+ spaces are no longer needed, or have been replaced by digital apps, which tend only to serve small sections of these communities".
More than half of London's LGBTQ+ venues have closed down in the past decade, according to a new report.