Pupils in S4-S6 at schools across central Scotland and Dundee will have to start wearing face masks in classrooms.
The new guidance also applies to their teachers at schools in level three and four areas under the new Covid alert system beginning on Monday.
Parents, meanwhile, will have to wear face coverings at drop-off and pick up at all schools in Scotland.
Deputy First Minister John Swinney said the changes were being made in response to “updated scientific and health advice”.
But the timing of the announcement was criticised by opposition politicians, while one parents group said the decision had been taken “on a whim”.
Under the new guidelines for levels three and four, teachers and senior pupils must also wear masks when moving around schools or in communal areas.
Level three includes Glasgow, Edinburgh the Lothians, Lanarkshire, Ayrshire and Dundee. No council areas are currently ranked in level four.
Meanwhile, adults in all schools must wear masks when they cannot keep more than two metres away from other staff, parents and pupils above P2.
New guidance for staff and pupils in the shielding category is also being issued by the Scottish Government ahead of a new five-tier system of restrictions designed to control coronavirus coming into force at 6am on Monday.
Swinney, also the education secretary, said: “From Monday, there will be increased use of face coverings and new advice to help protect those in the shielding category.
“It is vital that all the measures are followed rigorously in schools. Doing that – together with the collective efforts of all of us across wider society – will help to ensure that schools can safely remain open.”
Friday morning’s announcement was questioned by the Scottish Conservatives, who said it should have been debated in parliament.
Shadow education secretary Jamie Greene said: “Once again, significant Covid-19 restrictions are announced by the SNP government hours after parliament closes. Had they made this known on Thursday, this could have been properly debated.
“Parents across Scotland understood the guidance around staff wearing masks in schools, and older pupils wearing them in public areas, but the SNP has not explained why this latest move is necessary.
“Nor have they addressed legitimate concerns about pupils’ wellbeing, particularly for those with additional support needs.”
Parents campaign group UsForThem Scotland said its members wanted to see scientific evidence to support the decision.
Organiser Jo Bisset said: “It will be incredibly uncomfortable for young people to spend several hours a day in a mask, and could impact on their learning and education.
“We have particular concerns about the impact it will have on vulnerable pupils and those with autism or hearing difficulties.
“All parents ask is that, when such significant decisions are taken, the reasons and evidence for it are backed up.”
She added: “Parents suspect this decision has been made on a whim, and is merely just the start of an escalation which will eventually see all pupils forced to wear masks.”
Teaching union the Educational Institute of Scotland (EIS) said it supported the use of face masks, but insisted social distancing was impossible in classrooms.
General secretary Larry Flanagan said: “The strengthening of the school guidance in a number of areas is a reflection of the greater risk posed by higher levels of community infection.
“Whilst the EIS supports the stronger advice in areas such as the use of face coverings and the critical need for adequate ventilation, we remain dissatisfied with the lack of specification on social distancing between pupils, which is exhorted but remains impossible to achieve in full classrooms.”
The council areas in level three are: Inverclyde, East Dunbartonshire, West Dunbartonshire, Renfrewshire, East Renfrewshire, City of Glasgow, South Ayrshire, East Ayrshire, North Ayrshire, Stirling, Falkirk, Clackmannanshire, City of Edinburgh, Midlothian, West Lothian, East Lothian, Dundee City, North Lanarkshire and South Lanarkshire.