FA Cup defeat at Chelmsford City saw the former Southend United boss leave the EBB Stadium.
The Shots sit in the National League drop zone with just one win since early September.
Aldershot legend Terry Brown will take the team until a replacement is found.
More was expected of the club this season following a struggle at the foot of the table last term.
But a number of poor performances has seen a change at the helm as the Hampshire club plot a path to survival.
The club confirmed in a statement: “Aldershot Town Football Club can confirm that First-Team Manager Mark Molesley and Assistant Manager Tom Prodomo have been relieved of their duties with immediate effect.
“Terry Brown will take temporary charge of first-team affairs whilst the club begin the search for a permanent successor.
“We would like to thank Mark and Tom for their hard work over the last year and wish them well for the future.”