The Finance Secretary, who was narrowly defeated by Mr Yousaf in the SNP leadership contest, is understood to have been offered a demotion to the rural affairs brief.
Sources close to her confirmed she had told the incoming First Minister that she did not want the job and would instead be returning to the backbenches.
The incident immediately derailed Mr Yousaf’s attempts to draw a line under the bitter SNP leadership contest, which had resulted in the normally united party descending into infighting.
During the campaign, Ms Forbes publicly attacked Mr Yousaf over his record in government and said electing him would be an “acceptance of mediocrity”.
Alex Neil, the former Scottish health secretary who backed Ms Forbes during the contest, said Mr Yousaf’s job offer was “an insult and not a real effort to unite” the SNP.
A source close to Ms Forbes confirmed that she had turned down a position in Mr Yousaf’s administration but insisted that their conversation had been “very amicable”.
“Kate let him know she did not wish to be considered for a Cabinet position at this time,” they added. “Kate is now a mum with a young family, and Humza completely understands.”
Ms Forbes said on Tuesday that she was “delighted” to vote for Mr Yousaf as First Minister and that he had her “full support as he governs well and furthers the case for independence”.
“I have full confidence he will appoint a talented Cabinet and ministerial team, able to meet the challenges facing the country,” she added.
Scottish Conservative chairman Craig Hoy said Mr Yousaf had “poured petrol on the SNP civil war” immediately after being confirmed as First Minister.
“Kate Forbes’ furious snub to his offer of a Cabinet demotion shows his mission to reunite their feuding party is doomed to failure,” he added.
Mr Yousaf was confirmed as Scotland’s sixth First Minister in a vote at Holyrood on Tuesday, revealing afterwards that Shona Robison, one of Nicola Sturgeon’s closest allies, would be his Deputy First Minister.
He also confirmed he would have a “minister for independence” in his Government, a role he promised to create during the leadership campaign.
In his acceptance speech, Mr Yousaf said he had “lost count” of the number of times his identity as a Scot had been questioned due to his race.
“There was a time not that long ago when I felt I simply did not belong here,” he added, saying he hoped his election would give hope to others in a similar position.
He will be formally sworn into office at the Court of Session in Edinburgh on Wednesday before taking part in his debut First Minister’s Questions on Thursday.