The raptor, known as Lady, produced a record 69th egg at 12.30am on Sunday, on her nest at the Scottish Wildlife Trust's Loch of the Lowes reserve, near Dunkeld, Perthshire.
Staff and volunteers who watch over the nest recorded two males close to the nest site at around 2.15pm.
The bird was frightened from her nest, leaving her egg unattended for around 20 minutes.The intruders, who walked past signs to stay clear, were intercepted by wardens and police.
Emma Rawling, SWT's Perthshire ranger, said: "We did have a nest disturbance incident with humans. The birds were disturbed and frightened off the nest, leaving the egg exposed for about 20 minutes. Lady was frightened off the nest by their presence, and even when she returned she was too scared to sit down and incubate. She was really upset."
The 28-year-old female and her mate known as Laddie had been incubating the egg for just over 13 hours when the incident occurred.
Staff at the centre hope Lady will fledge a record 51st chick this season. A spokeswoman for Police Scotland Tayside Division said investigations are ongoing.
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