Plans were unveiled last month to build 27 additional rooms at Cromlix House and a new function suite and tearoom to cope with soaring demand.
Double Wimbledon champion Murray, 30, bought the property near his hometown of Dunblane for £1.8 million in 2013 and it has won a string of awards since reopening the following year.
However, surveys of the buildings earmarked for the extra rooms have revealed more than 130 bats roosting in the area.
Bats are a protected species under UK law and it is illegal to kill, possess or handle them without a licence or to cause them harm or disturb their habitat in any way.
An ecological report submitted to Stirling Council said: “Soprano pipistrelle bats were recorded roosting in the buildings on site during the nocturnal surveys. Common pipistrelle, Daubenton’s and Natterer’s bats were also recorded foraging on site, but no roost sites for these species were confirmed.
“Bat roosts were identified within all of the buildings on site, with the exception of a small, dilapidated portable cabin to the north.
“Many of the roosts sites supported one or two roosting bats; however, it is evident that the buildings within the square support a maternity colony of bats, with over 130 bats observed emerging from the buildings during the final nocturnal survey.”
The report states that the hotel will need to obtain a licence from Scottish Natural Heritage and implement mitigation measures
to protect the bats before work can be carried out.
It also recommends that access holes should be constructed on the roof of the newly refurbished buildings to allow bats to enter the loft space and bat boxes should be installed in the grounds during building work.
Work may also be banned in some areas during summer months as maternity roosts were found where female bats have their babies during that period.
Sue Sexton, of the Scottish Wildlife Trust Stirling and Clackmannanshire group, said: “It was encouraging to see that a thorough bat survey had been done as part of this application.
“The site is clearly important for at least two species of bat with a number of roosts including maternity roosts. We very much hope that the recommendations of the report will be included in the conditions of approval if approval is granted.”
Earlier this year Murray agreed to share his new mansion with bats in order to allow building work to go ahead.
The tennis ace is to allow the protected species to roost in the loft space of the new home he is having constructed.
Specially designed tiles will be erected on the roof to allow the creatures access to the property in Leatherhead, Surrey.
The planning applications for Cromlix House have gone out to public consultation and officials at Stirling Council are expected to make a decision on them by June.
At peak season a suite at the Cromlix costs £595 a night while a standard double room is £385. The restaurant is overseen by Albert Roux, the Michelin-starred chef. Afternoon tea costs £22.50 per person with a champagne option also available for an additional £10.
Cromlix has become the venue for Murray family gatherings. William Murray, Andy and Jamie Murray’s father, married his long-term partner at the hotel in 2016.
The 15 rooms and suites are named after some of Scotland’s most recognisable figures, including Sir Chris Hoy, Sir Alex Ferguson, Annie Lennox, Sir Arthur Conan Doyle and William Wallace.
The hotel was originally a private residence before being converted in 1981, a century after it was built.
Murray bought it in an effort to attract more visitors to the area around his home town and to boost the local economy.
It has twice won Scottish Hotel of the Year at the Hospitality Awards and earlier this year became one of only two Five Star Gold Country House Hotels in Scotland following a grading by VisitScotland. .