The pair took the reins at Arbour Park back in 2013, taking the club from Step 4 to the Vanarama National League South.
Form since mid-September has been poor - the Rebels have won just once since their FA Cup exit to Worthing.
Manny Williams, Lee Togwell and Scott Davies will take temporary charge while the club finds a replacement.
A statement released by the club on Monday evening confirmed their departure after nine-and-a-half years.
The statement read: "This evening, during a meeting with the club’s board of directors, Jon Underwood and Neil Baker informed the board of their intention to resign as joint first team managers of Slough Town Football Club with immediate effect.
"Naturally we are extremely disappointed at the news, which brings to an end Jon and Neil’s nine-and-a-half year tenure as joint managers since arriving in May 2013.
"Manny Williams, Lee Togwell and Scott Davies will take charge of tomorrow evening’s Berks & Bucks FA Senior Cup tie at Maidenhead United.
"A further statement will be issued in due course when the board have had more time to reflect on this development."