Left-back Peter Gilbert shot low into the bottom corner of Jose Veiga's goal from Ryan Hall's corner in the 24th minute to put the Shrimpers in the driving seat.
With promotion rivals Torquay losing 3-0 at Hereford and Crawley goalless at Accrington at the time, Southend moved into the third and final automatic promotion spot.
But Crawley midfielder Scott Neilson fired a 67th-minute winner at the Frazer Eagle Stadium to ensure his side reclaimed the third place they had held before kick-off.
Neil Harris snatched a second for the Blues in the 90th minute when Veiga parried substitute Elliot Benyon's header and the veteran striker put the rebound away, while Macclesfield put in a spirited performance but rarely threatened Southend's goal.
The scoreline meant Southend finished fourth and face seventh-placed Crewe in the play-off semi-finals.