Both John Lamont, who represents Berwickshire, Roxburgh and Selkirk and David Mundell, for Dumfriesshire, Clydesdale and Tweeddale were among the 148 MPs who cast their votes in favour of a change of leader.
Mr Johnson did win 211 votes from his Parliamentary party to ensure he remains in office but the closeness of the ballot has placed his long-term future in doubt.
After casting his vote Mr Lamont issued a statement on his Twitter account revealing his decision was made after he had consulted with his constituents.
He has also resigned as parliamentary private secretary to Foreign Secretary Liz Truss.
Mr Lamont said he had made the decision after “considerable thought and reflection”, adding: “I have consulted widely with my constituents in the Scottish Borders.
"I have also received thousands of emails and had countless conversations about the Prime Minister’s performance over the last few weeks and months.
“The events in Downing Street during lockdown were unacceptable. People across the country have rightly been deeply angered by what went on.
“Whilst I recognise the changes that have been made in Downing Street, the unfortunate reality is that the government has become overwhelmed by these events, to the detriment of my constituents and the people across the United Kingdom. The amazing work that this government is doing in Scotland and across the UK is increasingly being overshadowed.
“This has been an incredibly difficult decision. I know that many colleagues have been wrestling with their conscience during today and I respect that some MPs will have a different conclusion.”
Meanwhile, Mr Mundell, also on his Twitter account, issued a much shorter statement, saying: “After a difficult couple of years and listening to the views of my constituents, I voted for a fresh start and new leadership for our country.”
The two Borders MPs were among four Scottish Conservative MPs that have confirmed they voted against Mr Johnson, the others being Andrew Bowie, for West Aberdeenshire, and Scottish Conservatives leader Douglas Ross, MP for Moray.