Former Wales midfielder Fletcher, 32, had been in charge of the Pilgrims since September 2011 but this season has seen no improvement on last term's performance, when Plymouth finished 21st in the bottom tier of the Football League.
Tuesday's loss to second-bottom Rovers saw Plymouth's cushion above the relegation zone trimmed to two points, as they tumbled a place to again find themselves in 21st place.
Under their own new boss, John Ward, Rovers went ahead after 19 minutes through Joe Anyinsah and doubled their lead six minutes later when Matthew Lund netted.
Plymouth pulled one back when Conor Hourihane scored after 62 minutes, but it was not enough for them, or their manager.
Port Vale relinquished top spot in the table to Gillingham after suffering a 2-0 home setback against Fleetwood, who jumped two places to sixth after first-half goals from David Ball and Alan Goodall.
In a lunchtime game, Gillingham were 1-0 victors at Southend, their winner coming from Chris Whelpdale in the third minute.
Rotherham leapt two places to third, the final automatic promotion position, after coming from behind to prevail 2-1 at Rochdale.
Ashley Grimes put Rochdale ahead just before the half hour, but Kayode Odejayi levelled with 12 minutes to play and Courtney Cameron struck at the death.
The points guided Rotherham above Southend and also Cheltenham, who had previously held third spot but stumbled to a 1-0 loss at Oxford. Peter Leven's 16th-minute penalty made the difference at the Kassam Stadium.
Capital One Cup semi-finalists Bradford were held 0-0 at Morecambe and fell a place to seventh.
Burton also slid one spot, to eighth in their case, after losing 3-0 at mid-table York, whose goals came from Jason Walker, with a penalty, Patrick McLaughlin and Matty Blair.
Romuald Boco's goal gave Accrington a 1-0 success against Chesterfield and a late own goal from young defender Kortney Hause meant Exeter won by the same margin at Wycombe. Exeter had Jamie Cureton sent off in the first half and Wycombe's Joel Grant missed a second-half penalty.
Aldershot overcame fellow strugglers Barnet at Underhill, with Craig Reid's penalty in the 48th minute the only goal. Barnet had Jonathan Nurse sent off soon afterwards.
Stacy Long secured maximum rewards for bottom-of-the-table AFC Wimbledon from their trip to Torquay, his late goal bringing a 3-2 victory.
The Dons looked set for a straightforward win as they led 2-1 approaching the closing stages following goals from Paul McCallum and Mat Mitchel-King. Lee Mansell had netted for Torquay.
Torquay found a late leveller to make it 2-2 but Long had the final say in the fourth minute of stoppage time.
There was no such drama at Northampton where goals from Abu Ogogo, Chris Hackett and Kelvin Langmead for the home side rendered Scott Doe's reply in the 71st minute for Dagenham and Redbridge rather inconsequential.