Dominic Cummings has accused Carrie Symonds of trying to "appoint her friends to particular jobs" in a manner that was "completely unethical and clearly illegal".
He told a committee of MPs that his resignation last November was linked to Boris Johnson’s girlfriend who was trying to "overturn an official process of hiring".
And he added that "the heart of the problem" was he thought the prime minister was "unfit for the job".
Giving evidence to the Commons Health and Social Care and Science and Technology Committees Mr Cummings said: "My resignation was definitely connected to the fact that the prime minister’s girlfriend was trying to change a whole bunch of different appointments at Number 10 and appoint her friends to particular jobs.
"In particular she was trying to overturn the outcome of an official process about hiring a particular job in a way which was not only completely unethical but was also clearly illegal.
"I thought the whole process about how the prime minister was behaving at that point was appalling and all that was definitely part of why I went."
Earlier he said Ms Symonds was "desperate" to get rid of him.
He added: "In the summer I brought in Simon Case to be permanent secretary at Number 10 because I thought the prime minister is not listening to me on this whole subject [COVID], our relations are getting worse and worse, his girlfriend is desperate to get rid of me and all my team.
"If I bring in someone official to try to take over on COVID that will a, make things better, and b, maybe he will listen better to Simon Case than he will to me."