From the 2017-18 season, clubs which gain promotion to the Huws Gray Alliance - one level below the Welsh Premiership - must be able to provide 250 seats for supporters.
Glantraeth Football Club told S4C's Newyddion 9 it would rule them out.
The Football Association of Wales said the aim was to improve standards and grants were available.
Glantraeth chairman Gareth Evans said: "There aren't many people living in Glantraeth, there aren't 250 people who would come and watch us.
"A lot of the clubs on Anglesey won't be able to do it, it's impossible to do it."
Glantraeth FC, which has actress Naomi Watts as an honorary president, plays in the third tier Welsh Alliance football league division one and attracts crowds of about 40 people, with capacity for 63.
New guidelines could stop football clubs gaining promotion to higher leagues, a club on Anglesey has warned.