Scots returning from holidays in Portugal and French Polynesia will need to quarantine for 14 days when they return starting from Saturday.
It comes just two weeks after Portugal was placed on the exempt list.
The Scottish Government joined the Welsh administration in reinstating quarantine measures for Portugal, but the UK Government did not do the same for arrivals from the country to England.
In Portugal’s case, the risk of importation into Scotland by people who have visited the country “is too high to ignore”, the Scottish Government said.
Falling cases in the southern European tourist destination had seen travellers given an exemption from quarantine, beginning on August 22.
But it will have lasted just 14 days when self-isolation for all arrivals from Portugal to Scotland will be required from 4am on Saturday.
Portugal and French Polynesia are the latest countries to join the growing list of nations moved from the exempt to the quarantine list amid rising coronavirus cases in Europe and around the world.
In addition, Gibraltar is high on the Scottish Government’s watch list of countries that are being monitoring closely.
Failure to comply with requirement to quarantine can result in a fine of £480.
“We are in the midst of a global pandemic and the situation in many countries can change suddenly,” said Justice Secretary Humza Yousaf.
“Therefore, people should think very hard before committing to non-essential travel abroad.
“With Scotland’s relatively low infection rate, importation of new cases is a significant risk to public health.
“I would also encourage people who have returned to Scotland from Portugal or French Polynesia in the last few days to be particularly careful in their social contacts and to ensure they stick to the FACTS.
“I am also concerned by the level of infections in Gibraltar and we will be monitoring the situation there very carefully.
“Regular discussions continue with the other three governments in the UK. We continue to closely monitor the situation in all parts of the world and base the decisions we make on the scientific evidence available.
“The requirement for travellers to quarantine for 14 days on arrival from a non-exempt country is vital to help prevent transmission of the virus and to suppress it – not doing so poses a significant risk to wider public health across Scotland.”