After fives years of planning, legal challenges and protests from conservationists, the operation began shortly after sunset.
At 5.21pm, a cacophony of percussion noises could be heard from within the gardens, which were closed to the public.
A mix of industrial noises, whistles and what sounded like the banging of saucepans was relayed from speakers on mobile buggies.
Within five minutes of the wake-up call, hundreds of bats were circling above the gardens.
''Normally you get a stream flying to the south and one to the east but they are not doing that,'' said Storm Stanford of Bat Watch Australia. ''You can see how confused their flight is and they are making a lot of noise. Normally it is silent.''
The hope is that the grey-headed flying fox will disappear into the Sydney night skies and never return.
The biggest unknown, however, is exactly where the Pteropus Poliocephalus will choose for their new hang out. There are concerns that the bats, carriers of the Hendra virus, may add to the numbers at Centennial Park, close to Randwick racecourse.
''We will have a better idea how it has gone after a few days, it may even take a fortnight,'' the Royal Botanic Gardens and Domain director, Mark Savio, said. ''I think it may need to be a little bit louder. The idea is that you don't let them get used to a noise pattern.''
He said there was a small window of opportunity to act while the colony was at a seasonal low of about 5000.
The recorded noise is played for up to 45 minutes before dawn and 30 minutes around sunset.
The noise disturbance will be focused in the centre of the garden, near the Palm Grove.
Their roosting is blamed for the loss of 28 trees and 30 palms, including some irreplaceable heritage species. A further 60 plants are on the critical list.
''Strict approval conditions require that the flying foxes relocate to suitable locations away from homes,'' Mr Savio said. ''If they settle in unsuitable locations, we're committed to moving them on from there.''
In accordance with NSW and federal government approvals, an extensive monitoring and tracking study is supporting the relocation. The dispersal can occur only from May 1 to July 31 to ensure the flying foxes, which are classified as vulnerable, are not disturbed during later pregnancy, when the young are being born and when mothers are carrying young.
''It will be like musical chairs, when the music stops that's where the foxes will be. There's no basis for telling whether the animals will be in an acceptable spot on July 31,'' the flying fox researcher Peggy Eby said previously.