She ended the bid after doctors warned that another sting from a Portuguese Man-Of-War could be life-threatening.
The 62-year-old had swum about 49 miles (79km) in shark-infested waters after setting out from Havana on Friday.
The Los Angeles woman was trying to break her own record for open-water swimming without a shark cage.
Her first attempt at the swim, in August, had been cut short by an asthma attack.
Her website says she decided to end her latest swim at 11:00 on Saturday (15:00 GMT), after swimming for more than 40 hours.
"The medical team said I should not go another two nights in the water and risk additional likely Man-of-War stings which could have a long-term cumulative effect on my body," Ms Nyad says.
"But for each of us, isn't life about determining your own finish line? This journey has always been about reaching your own other shore no matter what it is, and that dream continues."
Ms Nyad first attempted the Cuba-Florida crossing, inside a steel shark cage, in 1978 when she was 28, but heavy seas forced her to give up.
A 22-year-old Australian, Susan Maroney, completed the swim - in a shark cage - in May 1997, Reuters news agency reports.
Ms Nyad had said she hoped the swim would inspire others to lead active lives as they age, as well as help foster an increased understanding between the US and Cuba.
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