Jessica Gould, who has been at the garden 11 years, and Laura Davies, who joined the staff eight years ago, have said that it is "time to move on" to new challenges.
Ivor Stokes, a previous director of horticulture at the botanic garden, had agreed to step in on an interim basis while the recruitment process for a permanent post-holder is progressed.
Garden director Kevin Lamb left suddenly two months ago and the garden has yet to appoint a permanent successor.
Chairman of trustees Rob Jolliffe said: "We are very sorry to see these ladies leave us. They have done an enormous amount of very hard work for the Garden and we owe them a great debt of gratitude.
"They will be missed and we wish them well in whatever the future holds for them."
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