In response to the offer, the entertainment firm said it had full confidence in Eisner's strategy for the firm and would consider any "legitimate proposal".
It dismissed the Comcast offer by saying that it did not best serve Disney shareholders.
In a statement Disney said: "The board has confidence in the business, financial and creative direction of Disney under the leadership of Michael Eisner and his management team. The interests of Disney shareholders, which represent the fundamental priority of the board, would not be served by accepting any acquisition proposal that does not reflect fully Disney's intrinsic value and earnings prospects."
Comcast has responded to the rejection by saying that it has no plans to give up the fight and added that its bid for Disney was "compelling" and "sound".
"Our proposal to acquire the Walt Disney Company reflects a full and generous valuation based upon Disney's prospects and performance over a long period of time, representing a significant premium over Disney's unaffected share price during any relevant measurement period over the last three years," Comcast said.
Eisner was fighting for his corporate life last week after the Comcast bid was made. His strategy has been under fire and there was speculation that he would not survive.
The solidarity of the Disney board behind Eisner surprised many. Eisner had been under attack for not understanding the animation business and for overspending on acquisitions in its theme-park operation, which is faring badly in a post 9/11 world.
One of the problems for Comcast is that its deal is an all-stock one and its share price has fallen. Originally valued at $54bn (£28.9bn) the deal sank to less than $48bn in Comcast stock on Friday. There was no trading yesterday, the US President's Day holiday, but Comcast argued yesterday that its offer was still fair.
"We maintain the belief that our merger proposal represents a sound and compelling proposition for both sets of shareholders," Comcast said.
Analysts were predicting that Comcast could sit tight and wait for its share price to rise while Disney could lose ground. It is thought unlikely that rival bidders will emerge. MTV and CBS owner Viacom has already ruled itself out.
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