The video, featuring young Charlie biting the finger of his older brother Harry, has been viewed 500,022,774 times since the boy's father uploaded it in May 2007.
Changing consumption of Charlie bit my finger - again! gives a snapshot of shifting habits in online video streaming over the years, with the ever increasing adoption of internet connected mobile devices.
Around two thirds (73.5%) of all views of the video in 2012 came from smartphones and tablets, compared to 45.2% in 2011 and 22.4% in 2010, according to data supplied by Rightster.
The UK-produced video has gained a worldwide audience since it was posted, attracting views in the USA, Mexico, Canada, Germany, Brazil, Australia and most recently, Saudi Arabia.
There have been 103 views from the Vatican City and three views from Tokelau, a tiny of New Zealand with a population of approximately 1,400 people.
Charlie bit my finger - again! was initially more popular among females, at over 60% of the viewing audience in 2007, but its audience is now broadly split between males and females.
Howard Davies-Carr, the father of Harry and Charlie, said: "We love that such a simple video has touched so many souls. We hope the boys can continue to lighten up peoples' lives in the years to come."
The half a billion milestone came as new videos featuring Harry and Charlie were posted on YouTube, also introducing their younger brother Jasper.
Produced by Viral Spiral in conjunction with Rightster, the pilot episodes include Biting News, King of The Week and Charlie Bites…Toys.
"We're delighted that the Charlie Bit My Finger clip has now reached the half a billion views milestone - it's amazing to see how popular this video has become in every corner of the globe," Damian Collier, the founder and chief executive of Viral Spiral said.
"It's a household name, and turning into a true 'brand'. We look forward to working with the boys and their family as well as Rightster, on this exciting new venture."
Rightster founder and chief executive Charlie Muirhead added: "The immense popularity of the Charlie bit my finger video underscores the massive potential for content producers, publishers and brands to create, distribute and monetise original video content and demonstrates the potential to extend their reach to millions of online users globally."
Last month, 'Gangnam Style' by South Korean musician Psy became the most viewed video on YouTube of all time, attracting more than 805m views and beating Justin Bieber's song 'Baby'.