The strife-torn tourists were straight back into turmoil with enigmatic opener Dave Warner sent packing to an awful shot in just the second over.
Hughes, 24, was victim to a vicious over from Ishant Sharma before he chopped a ball onto his off-stump.
Australia went to lunch at 2-94 with Hughes gone for 45 and reliable opener Ed Cowan in stout resistance on a patient 27 from 83 balls.
Stand-in captain Shane Watson was 16 after he had joined Cowan at the loss of Hughes.
Watson survived a horribly nervous third-umpire review just 10 minutes from the lunch break when he was millimeters from being stumped by opposite captain Mohendra Singh Dhoni from left-hand spinner Pragyan Ojha.
Watson was given benefit of doubt when his back foot was planted on the popping crease.
He was on seven and Australia 2-80 in the 25th over and then belted two confident boundaries off Ojha in the last over before the break.
Hughes was roughened from a ferociously lifting ball from Sharma that smashed into the Aussie batsman's face grill on his helmet.
A gritty Hughes was out three balls later.
Warner slashed at a wide and angled delivery from big Ishant Sharma and edged a straight-forward catch to Verat Kohli at second slip.
With Warner out the beleaguered Hughes set about salvaging the innings alongside trhe steady Cowan.
Hughes raced through the 20s on his arrival at the crease with seven fours from just 23 balls in his first 29 runs.
Hughes had been on the brink of being dumped after scores of 6, 0, 19 and 0 from the first two Tests of the series but resurrected his number three spot with a feisty 69 in the second inings at Mohali when replacement candidates had been banned from playing.
It was another irrational shot from the attacking Warner so early in an important innings.
Watson had an early minor victory when he won the toss and elected to bat.
It was the fourth successive toss won by the Aussies but the tourists had been thumped in all three previous Tests.
Watson slotted in at number four in the revamped Aussie line-up attempting to avoid an embarrassing four-nil Test series loss for the first time since 1970 against South Africa in South Africa.
Cowan and Hughes had each opened their account with boundaries in the off-side, before Sharma and impressive young paceman Bhuvneshwar Kumar proved awkward to handle with early signs of malpractice from the dry and cracked pitch.
The home unit brought series leading wicket-taker Ravi Ashwin into the attack after just eight overs.
Australian Test superstar Shane Warne labelled Warner as "irresponsible" for his loose shot outside off stump and so early into his innings.
"It was a bit 'wishy-washy' and loose. Australia needed something better from Dave Warner," Warne said on Fox Sports coverage of the opening overs.
Watson stepped in as Aussie skipper with trump batsman Michael Clarke forced to miss the Test with a nagging back injury.
Watson is among five changes to the embattled Aussie unit with pacemen James Pattinson and Mitchell Johnson as well as spin bowling all-rounder Glenn Maxwell also recalled.
Regular wicketkeeper Matthew Wade returned after recovering from a sprained ankle, replacing veteran Brad Haddin who played as fill-in gloveman in Mohali.
Pattinson and Johnson are in for big left-arm seamer Mitchell Starc and struggling spinner Xanvier Doherty.
Maxwell replaces Moises Henriques who was dropped after two successive poor Tests following an outstanding debut in Chennai when the all-rounder hammered 68 and 81 not out.
Johnson is the 16th player Australia has played in the four-Test series.
It is only the third time in Australian Test history that more than 15 players have been used during an overseas tour.
The Aussies used 17 players on the 1983-84 tour through the West Indies and 16 played on the 1985 series in England.
India was forced to replace Third Test debut sensation Shikhar Dhawan, who plundered a history-making 187 from just 174 balls at Mohali last weekend.
The stroke-making left-hander was replaced by Ajinkya Rahane at the top of the home nation's batting line-up.
Rahane, 24, has played 16 one-day internationals but not a Test before his call-up to replace Dhawan, when he did not recover from a hand injury he suffered while fielding in Australia's second innings at Mohali.