Patricia Laing gifted £100,000 to the zoo, with the express wish it be used to care for its world-famous penguins and chimps.
Miss Laing, of Edinburgh, died aged 96, in March this year. Her published will revealed she had amassed an estate worth £3,792,790.79 by the time of her death. The retired clerical assistant had never married and lived in Edinburgh all her life. She moved into a care home in the capital in her later years.
The bulk of her fortune was left to good causes, with 31 separate legacies to be handed over to charities.
But Miss Laing asked that money be given to the Royal Zoological Society of Scotland and used at Edinburgh Zoo "for the care of animals, especially the penguins and chimps".
The zoo first opened its gates in 1913, and is home to the UK's only pair of giant pandas. Its penguin parade - which is now a much-loved daily occurrence - came about by accident in the 1950s after a zookeeper mistakenly left the gate to the penguin pool open and was followed around the site by a gang of penguins.
The spectacle went down so well with visitors it became a regular feature.
Miss Laing left almost £90,000 in gifts to friends but asked that the remaining cash be split up between charities including Macmillan Cancer Research, the Multiple Sclerosis Society, and Guide Dogs for the Blind, Oxfam, the National Asthma Society and the British Lung Foundation.
They will now be in line to receive around £117,000 each.
Dana O'Dwyer, chief executive of Capability Scotland, which also received a legacy from Miss Laing, said: "We are very grateful to Miss Laing for remembering us so thoughtfully in her will. Her generosity, and the generosity of others like her who leave legacies to Capability Scotland, will help ensure our continued support of disabled people at school, at home and at work."
Miss Laing's will showed she had built up a substantial stocks and shares portfolio by the time of her death. She had a £2.6m investment with financial group Investec Wealth and Investment and also had almost £900,000 in savings and ISAs.
Her will shows her jewellery was valued at £520 and she had £1117 held in a Clydesdale Bank account in Edinburgh.
A Guide Dogs Scotland spokesman said: "Legacies and gifts in wills are really important to Guide Dogs Scotland as they fund two out of three guide dogs.
"It costs around £50,000 to train and support each guide dog throughout its working life, and this is supported entirely by public generosity.
"Legacies and gifts in wills are an invaluable contribution to our life-changing service."