The Red Badgers went into Easter in a three-way battle for the title but, despite a 3-2 away win at Gresley Rovers on Saturday, they lost 3-0 at home to Belper United on Easter Monday.
That meant Lincoln United were crowned champions with Bourne Town second and Eastwood third, three points short having been top of the table for much of the campaign.
They now host Boston Town in the play-off semi-finals this Saturday.
Joint managers Martin Ball and Daryll Thomas said: “Obviously, we’re massively disappointed to miss out right at the end.
“It’s been a long, hard season and, in the end, it came down to one poor performance - unfortunately, at the worst possible time.
“We always knew we needed results elsewhere to go our way, but first and foremost, we had to take care of our own business.
“Sadly, we weren’t able to do that, and we’re absolutely gutted.”
Reflecting on the season as a whole, they added: "We’ve been up there all year, fighting every step of the way, and we’ve come so close to achieving our goal.
“To fall short like this is tough to take, but we’re incredibly proud of the group.
“The challenge now is to pick the lads up, regroup, and go again in the play-offs.
"There’s still everything to play for.”
At Gresley, goals from Aaron O'Connor, Tyler Blake and Oliver Robinson had Eastwood 3-0 up at the break.
But after Phil Watt was red-carded for an elbow on 53 minutes Gresley rallied and netted two late goals in a nervy finish.
It was then landlords versus tenants on Monday and Belper made themselves at home with goals by Keaton August (20 and 57 minutes) and Daniel Odunaiya (42) to stun the side they groundshare with.
But it was a good weekend for neighbours Kimberley MW who retained their Premier North status.
A 1-0 home defeat by Ashby Ivanhoe on Saturday had nerves shaking, but they hit back with a fantastic 3-0 away derby win at Hucknall Town on Monday thanks to goals by Ryan Wheatley, Oliver Seeby and Aarron Coyle, which saw them leap up the the safety of 17th place and Sleaford Town relegated.
Leading 3-0 they had Shea Thompson-Harris sent off after 63 minutes but saw it out.
After 22 games Kimberley were 12 points adrift at the bottom with one win and four draws and no wins in 20 games, but a surge of nine wins and draw in the last 16 games ultimately saw them safe.
However, Selston's relegation from Division One was confirmed in a 3-0 midweek defeat at Clay Cross Town and they signed off on Saturday with a 6-1 home hammering by Radford.