Oldham's 1-0 win over Yeovil meant that neither Pools nor Pompey can catch the Lancashire side.
It is all over for Portsmouth and Hartlepool, both of whom are eight points adrift of 20th place with just two matches left.
Pompey, winners of the FA Cup just five years ago, will now play in the Football League's bottom tier next season after suffering back-to-back relegations.
Matt Smith scored the only goal at Boundary Park as Oldham boss Lee Johnson secured bragging points over his father Gary in the Yeovil dugout.
The striker netted on the stroke of half-time to move the Latics five points clear of the drop zone with a game in hand over Colchester and Scunthorpe United, who occupy 21st.
Scunthorpe could be relegated at the weekend after losing 2-1 to Bury. Karl Hawley gave the home side the lead but the relegated Shakers hit back with second-half goals from Jonson Clarke-Harris and Nick Ajose to leave Brian Laws's men on the brink.
Colchester are the side United will have to catch to avoid the drop and they were beaten 2-0 by MK Dons after goals by Daniel Powell and Patrick Bamford.
At the other end of the table promotion-chasing Sheffield United and Brentford shared an eventful 2-2 draw at Bramall Lane. Marcelo Trotta gave the visitors the lead from the penalty spot but the Blades were handed the chance to equalise as Tony Craig was sent off after conceding a spot kick when he brought down Dave Kitson.
Tony McMahon missed the penalty but Barry Robson was not so generous as he netted from the spot after Lee Hodson had handled in the area.
Dave Kitson then put United ahead but the home side went down to 10-men as Harry Maguire was dismissed for a second yellow card and there was more drama to come as Clayton Donaldson was dismissed for a second booking before Bradley Wright-Phillips snatched a point for the Bees at the close.
In the night's other game two goals from Adam Rooney helped Swindon to a 4-1 victory over Crewe.