A group of 500 church leaders from around the UK have written to Nicola Sturgeon calling for her to lift the ban on public worship on Sundays.
Some 200 leaders from Scotland and another 300 from the rest of the UK say the decision may be unlawful.
As part of the latest lockdown rules, places of worship are only permitted to conduct weddings or funerals – with the number of people attending these strictly limited – and to broadcast services online.
Communal worship can continue south of the border subject to restrictions on attendance.
Last week, national clinical director Jason Leitch told Holyrood’s Covid-19 Committee that the ban on worship in-person was the right move.
He said the latest contact-tracing data showed a total of 120 Scots attended church or other places of worship while infectious with coronavirus over a seven-day period.
Signatories to the letter from several different denominations include Rev Paul Rees of Charlotte Chapel in Edinburgh and Rev Dr William Philip of the Tron Church in Glasgow.
The church leaders’ letter says: “We understand entirely the exceptional difficulties of leading the country at the present time, and we and our churches have prayed for wisdom and clarity for your government repeatedly.
“But we strongly disagree with the decision to prevent the gathering of the church at this time, which we believe is profoundly unhelpful and may be unlawful.”
It continues: “We know of no evidence of any tangible contribution to community transmission through churches in Scotland; to the contrary, since churches re-opened in July we have demonstrated that places of worship and public worship can be made safe from Covid transmission.
“It is for such reasons that legal challenges in other jurisdictions have overturned prohibitions of the freedom to gather for worship.
“However, above all we are dismayed because there seems to be a failure in the Scottish Government to understand that Christian worship is an essential public service, and especially vital to our nation in a time of crisis.”
A Scottish Government spokeswoman said: “We know how tough this decision will be for many people.
“However we must take action across society to stop the virus spreading further, so we can protect public health and save lives.
“This virus can spread anywhere that there is close human contact – without exception.
“Test and Protect tells us where people were in their 48-hour infectious period.
“So we know that on one day last week the seven-day number for places of worship was 120, and data from yesterday, Monday January 11, shows the seven-day number for places of worship is 38, underlining the essential decision to require places of worship to close for public health reasons.”