The 38-year-old, who first won the title in 1998, carded a three-under final round of 69 to finish 13 under, one and two shots clear of compatriot John Senden and Woods respectively.
Woods recorded an eagle and five birdies in a fine 67 as he battled back from a third-round 75.
Victory at The Lakes would have been a first for Woods in nearly two years.
The tournament ended in thrilling style, with Chalmers forced to watch nervously from the clubhouse as overnight leader Senden went close with a 35-foot birdie putt at the 18th that would have forced a play-off.
An emotional Chalmers, who fought back tears after beating a field including eight of the world's top 20, said: "I'm just extremely excited and extremely pleased to win this tournament twice.
"You can stumble into it once but to win it again, in this field, one of the strongest fields we've had in Australian golf for a long time, that does something for me, that really floats my boat.
"I think I stumbled into it when I was 26 ... I know what it takes now, it's hard to win."
A day shy of the second anniversary of his 95th and last title win at the 2009 Australian Masters, the crowd was thrilled as 14-time major winner Woods surged back from six shots behind.Woods last won a major at the US Open in 2008
Woods, 35, said: "A couple of drives on the back nine cost me. I fought real hard all day but I've come up just short.
"I'm just going to keep plugging along. It's just nice to be playing and not on the sidelines."
Western Australia-based world number 215 Chalmers started the day with birdies on his first two holes and held at least a share of the lead for the remainder of his round.
The left-hander stuttered with two bogeys before the turn but he sandwiched a birdie between them and two more in a flawless back nine which put him in the clubhouse with a two-shot lead.
Chalmers said he had spotted Woods moving up the leaderboard and heard the roar ahead of him when the American chipped in for an eagle at the 14th.
"I was aware of what Tiger was doing but I also knew I had birdie holes coming up," he said. "I thought he would have to make another eagle or birdie one of the harder holes."
Senden made a calamitous start with four bogeys in his first seven holes and his hopes of a second title to go along with his 2006 triumph looked all but gone at the turn.
But he stormed back with four birdies over the back nine, and having missed his putt on the final green by inches, finished with a 72.
Defending champion Geoff Ogilvy shot the best round of the day with a 65 to take a share of fourth place on nine under with compatriots Adam Scott (68), Jason Day (74) and Nick O'Hern (72), as well as American Nick Watney (72).