The 18-year-old stormed home to win in a junior world record of 47.58sec, 22-tenths of a second ahead of Belgian silver medallist Pieter Timmers' 47.80 with 2012 gold medallist Nathan Adrian of the United States third in 47.85.
Meanwhile Aussie sprint star Cameron McEvoy, who established himself as the man to beat with a sizzling 47.04 in April, was relegated to seventh in 48.12sec.
It was an electrifying Olympic debut for Chalmers, who ducked under the 48-second mark for the first time in Tuesday's heats.
"I actually had no idea about that," Chalmers said, adding that he was more inclined to follow basketball and soccer than swimming as a spectator.
Chalmers made the turn in seventh as Italy's Santo Condorelli led ahead of Adrian.
But he made his move along with Timmers, who was fifth at the 50m mark to seize the win in a mass finish.
As Chalmers put Australia back where they've long felt they belonged on the podium, Dmitriy Balandin was putting the swimming on the map in his home country -- making Kazakhstan's first Olympic medal in the sport a gold in the 200m breaststroke.
American Josh Prenot, who came into the Games atop the 2016 world rankings, was second in 2:07.53 and Russia's Anton Chupkov was third in 2:07.70.
Spain's Mireia Belmonte-Garcia rallied to win the 200m butterfly, improving on the silver she earned in London.
She caught front-running Australian Madeline Groves at the 150m mark, and finally got past her to touch in first in 2:04.85, just three-hundredths of a second in front.
Reigning world champion Natsumi Hoshi of Japan clocked 2:05.20 to match the bronze medal she claimed in London.
Allison Schmitt, Leah Smith, Maya DiRado and Ledecky clocked 7:43.03 to grab the gold ahead of Australia's Leah Neale, Emma McKeon, Bronte Barratt and Tamsin Cook, who touched in 7:44.87. Canada's Katerine Savard, Taylor Ruck, Brittany MacLean and Penny Oleksiak earned bronze in 7:45.39.
Michael Phelps continued his quest for a fourth straight 200m individual medley gold, besting old foe Ryan Lochte in their semi-final to lead the way into Thursday's final, where Brazil's Thiago Pereira and Japan's Kosuke Hagino will be out to crash the US duel.
Admittedly still tired, Phelps was nonetheless looking forward to a final showdown with world record-holder Lochte, who has taken two silvers and a bronze behind Phelps in the last three Olympics.
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