Rooted to the bottom of the third-tier standings, Duncan Ferguson's side needed to win and hope that Oxford United failed to beat Bolton Wanderers to stay alive.
The U's 1-0 defeat to Bolton gave Forest Green a slice of hope, but the Green were thrashed by Barnsley to go back down to League Two with a whimper.
Slobodan Tedic, Adam Phillips (2) and James Norwood put the game to bed before the hour mark, and Devante Cole rounded off the scoring for the Tykes after Reece Brown's consolation for Forest Green.
Ferguson's side have accumulated just 26 points from their 42 games this season, and 20th-placed Oxford hold an unassailable 14-point advantage over the soon-to-be League Two outfit.
Forest Green were promoted to League One for the first time in their history last season, winning the League Two title with 84 points to their name, pipping Exeter City to first place on goal difference.
Title-winning coach Rob Edwards departed for Watford in acrimonious circumstances before the start of the campaign, though, while Ferguson arrived in January to replace the outgoing Ian Burchnall.
With their fate sealed, Forest Green now prepare to take on Fleetwood Town, Cheltenham Town and Oxford before rounding off a doomed season against fellow relegation candidates Cambridge United.
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