Icahn, who also disclosed a 2.15 percent stake in the e-commerce giant on Monday, said in a letter to shareholders that two long-time board members, Marc Andreessen and Scott Cook, had business interests that directly competed with eBay.
The activist investor said he had found "multiple lapses in corporate governance" and called on both Andreessen and Scott to resign from eBay's board. Icahn also urged investors to vote for his two board nominees.
EBay in turn accused Icahn of "mudslinging" and said in a statement that he was attacking two "impeccably qualified directors." Representatives for Scott Cook and Marc Andreessen were not immediately available for comment.
Shares of eBay were up 3.5 percent at $56.49 in afternoon trading on the Nasdaq on Monday.
"If he's increasing his stake, and being more aggressive, it increases probability that he actually gets something done," Robert W. Baird & Co analyst Colin Sebastian said of Icahn.
Icahn's latest attack on eBay follows a major payday from his investment in Forest Laboratories Inc, where he was also pressing for a change in strategy and leadership. Forest announced last week that it would be bought by Generic drugmaker Actavis Plc for about $25 billion.
Andreessen is one of Silicon Valley's most prominent venture capitalists, while Scott is founder, former CEO and current board member of Intuit Inc, the maker of TurboTax and other business and personal financial software.
Icahn said Andreessen, who also sits on the board of Facebook Inc, had invested in and actively advised five direct competitors of eBay - Boku, Coinbase, Dwolla, Jumio and Fab. Four of these, Icahn said, were direct rivals to PayPal.
Icahn also took issue with the sale of Skype in September 2009 to an investor group that included Andreessen at a price that was 70 percent less than what eBay had paid. (http://link.reuters.com/zad27v)
The investor group sold Skype to Microsoft Corp for $8.5 billion about 18 months later, netting about $4 billion, Icahn said, questioning of Andreessen's loyalty.
Others have also been critical of the deal, Icahn said, "but, until now, none have taken on the task of standing up to Mr Donahoe and this board."
"How is it possible to engage in meaningful discussions about long-term stockholder value while ... the CEO seems to be completely asleep or, even worse, either naive or willfully blind to these grave lapses of accountability and stockholder value destruction?" Icahn said in the letter to shareholders.(http://www.shareholderssquaretable.com/open-letter-to-ebay-stockholders/)
EBay refuted the accusation by saying Andreessen had recused himself from decision making related to the Skype deal. It also said Donahoe was a "widely respected" CEO.
Andreessen, who is not up for re-election this year, has been on eBay's board since 2008, while Cook has been a director since 1998.
Icahn also took issue with Andreessen's investment in online shopping platform Kynetic, which eBay had sold back to its founders at a "low sale price" in 2011. Andreessen later invested $150 million in the company, for a paper gain of more than 100 percent a year later, Icahn said.
EBay said it had sold its stake in Kynetic at a profit.
Icahn also raised concerns about eBay's board having input from Cook when Intuit's Go-Payment service and PayPal were "direct competitors" in online payments processing.
Icahn noted that according to a Department of Justice complaint, eBay has agreed not to hire Intuit employees to placate Cook who had complained about eBay's hiring practices.
"Is Mr Cook wary of how a standalone PayPal could impact the company he founded? Is he worried that it would diminish the value of his $1 billion in Intuit stock?" Icahn asked.
EBay said the overlap with Intuit was small.
EBay bought PayPal for $1.5 billion in 2002 and has considered hiving off the multibillion-dollar division. But the company said last month that it had determined that PayPal would lose synergies with eBay's overall e-commerce business if it was independent.
On Monday, eBay reiterated that shareholders were better off if the PayPal remained part of the company.
Donahoe has been CEO of eBay since March 31, 2008. EBay's shares, which closed at $54.59 on the Nasdaq on Friday, have nearly doubled over that period, outperforming the 85 percent rise in the Nasdaq composite index.
The eBay board also includes Ford Motor Co Executive Chairman William Ford Jr and eBay founder Pierre Omidyar.
Soon after it was revealed last month that Icahn had taken a stake in eBay, Andreessen and Omidyar tweeted that they fully supported keeping the company intact.
"If he's right, then attacking the integrity of the (eBay) board is a tactic that will help him achieve his ends," said Paul Hodgson, an independent corporate governance analyst, referring to Icahn's accusations.
(Additional reporting by Chris Peters in Bangalore; Editing by Ted Kerr and Tiffany Wu)
By Nadia Damouni and Phil Wahba