Chesterfield have parted company with Martin Allen following the 4-0 defeat at home to Solihull Moors on Boxing Day.
Assistant manager Adrian Whitbread and head of recruitment and goalkeeping coach Carl Muggleton have also departed the club.
The 4-0 loss to Solihull saw the Spireites drop into the National League relegation zone, one point behind Dover Athletic in 20th.
The sacking of Allen comes after only seven months in charge of the Blues, earning just four wins from 25 league games.
Early statements from the former Barnet manager enticed fans and had them dreaming of promotion straight back to the Football League, and three wins out of the first three league games provided the start of a seemingly great season.
However, a run of 19 league games without a win and two consecutive defeats by Leyton Orient and Solihull Moors proved to be the final straw for Allen, Whitbread and Muggleton.
Newly appointed CEO Graham Bean told the club's website: "On behalf of the club, I would like to thank Martin and his staff for their hard work and wish them all the very best for the future. The search for a new manager is now underway."
John Pemberton, who became the club's new academy manager in September, has been put in charge for the game at home against Hartlepool United on Saturday.