The former Exeter striker came off the substitutes' bench to score twice inside ten minutes and keep the Pilgrims' play-off drive alive.
His first goal came within 11 seconds of replacing Jermaine Easter in the 75th-minute when he converted from close range after Steve MacLean's clever flick.
Five minutes before the end Mackie, who joined Argyle for £145,000 from their Devon non-league neighbours Exeter City in January, turned slickly in the area and fired past Tykes' goalkeeper Tony Warner.
His quick double finished off Barnsley, who had been reduced to ten men as early as the fourth minute when centre-back Lewin Nyatanga was sent off.
The Welsh international was red-carded for hauling back country-man Easter as the Pilgrims' striker latched on to MacLean's flick on from Lilian Nalis' through-ball.
Referee Andy Woolmer acted after consulting with linesman Andy Bennett, who broke his flag as he vigorously waved for an offence that it initially seemed Woolmer had missed.
To make matters worse for Barnsley, record £500,000 signing MacLean converted the subsequent free-kick with a stunning drive that gave Warner no chance.
Barnsley sacrificed striker Danny Nardiello and limited Argyle to just one clear chance before the interval, when home debutant Jim Paterson fired just over.
The visitors came more into the game after the break and Everton loan player Anderson De Silva smacked a shot that hit the crossbar and bounced down before being cleared, while Dennis Souza's composed far-post header from a corner also went close.
After Mackie's double strike, the icing on the cake for an ultimately comfortable victory for Argyle was the reappearance of club captain Paul Wotton after a lengthy injury absence.
Wotton came on to a standing ovation as a late substitute for his first first-team start since damaging his knee ligaments in a game against Hull in December 2006.