The luxury Auchterarder venue is working with NHS Tayside to identify the cause of the cases of vomiting and diarrhoea and put in place infection-control measures.
Most of the cases only came to light after guests who had stayed there last weekend contacted the hotel after falling ill.
Some staff and hotel residents are still recovering, but an NHS spokeswoman said nobody had been taken to hospital.
A spokeswoman for Gleneagles said a “mix” of residents, guests and staff were affected.
She said: “We are working with NHS Tayside. A number of cases were reported from guests last weekend, many of whom contacted us after they left.
“A handful are still not completely recovered. There are a few residents and staff who are still recovering.”
The hotel is home to Scotland’s only two-star Michelin star restaurant, Andrew Fairlie at Gleneagles. The prestigious hotel has played host to major golf tournaments and the international G8 summit in 2005.
It will also host the Ryder Cup golf tournament in 2014.
Mr Fairlie said none of his staff were affected and said the incidents were limited to the golf clubhouse, the Dormy.
The clubhouse overlooks the 18th holes of both the King’s and Queen’s courses and has recently undergone a “major renovation”, according to its website.
A spokesman for NHS Tayside said they were investigating the cause of the outbreak.
“NHS Tayside Health Protection Team and Environmental Health Officers from Perth and Kinross Council are investigating a number of cases of suspected norovirus in people connected to the Gleneagles Hotel. More than 100 people have reported symptoms over the past week,” he said.
“Staff at the hotel are working closely with NHS Tayside and Perth and Kinross Council to implement all the recommended infection-control measures.
“As always, anyone suffering from similar symptoms should be very careful with their hand hygiene and should remain at home until 48 hours after their symptoms have cleared up.”
Norovirus, commonly known as the winter vomiting bug, is the most common stomach illness in the UK, affecting between 600,000 and one million people each year.
Although most people make a full recovery within days, the virus forces hospital wards to close because of the danger to patients with weakened immune systems or compounds other illnesses getting treatment.
The virus can be spread through hand contact, but also through contaminated food or drink, or contact with contaminated surfaces or objects.
A Gleneagles spokeswoman added: “The care of our guests and staff is of paramount importance and we have been taking the necessary precautions. We are glad to say that as far as we are aware, only a handful of people have not yet returned to full health.”
Meanwhile, Thompsons Solicitors, which is representing those affected, said is had set up an emergency hotline. A spokesman said: “Outbreaks of gastric illnesses such as this can not only ruin holidays, they can also have serious health consequences.”