David Bowie received a standing ovation as he accepted the award for "setting the standard for how artists interact with their fans online".
EBay's award was dedicated to its 233 million users.
Picking up the award, CEO Meg Whitman told the audience: "Bidding starts at 99 cents."
Message for Alan
Now in their eleventh year, the Webbys allow recipients just a five-word acceptance speech.
The BBC News website highlighted the plight of kidnapped BBC Gaza correspondent Alan Johnston, saying: "Alan, we're thinking of you."
The BBC received the most nominations of any organisation in this year's awards.
BBC News Interactive Editor Steve Herrmann said: "To win both the Webby and the People's Choice awards again this year is a huge honour for us."
The Guardian, which won best newspaper website, also called for the release of Mr Johnston, who disappeared on 12 March.
British success in the news category prompted the host, US comedian Rob Corddry, to say: "The Brits are taking over."
User interactivity
YouTube founders Steve Chen and Chad Hurley won the award for Person of the Year.
They were presented with the award by online actress Jessica Lee Bowes, who played troubled teen Lonelygirl 15 in a fictional video diary on the site.
Their speech read: "You Tubers, this is for you."
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Alan, we're thinking of you
BBC News website message to reporter Alan Johnston
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They added: "We never thought so many things could be blended."
Artists of the Year were the Beastie Boys.
Their concert video, filmed entirely by 50 members of their audience, was held up as a shining example of user interactivity.
They asked the audience in New York's financial district: "Are we getting paid for this?"
They also thanked fans for their support.
The Webbys honour the best websites of the year in more than 100 categories.
The winners were chosen from nearly 8,000 entries from 60 different countries.
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