Di Matteo's biggest selection gamble since he took charge backfired as goals from Fabio Quagliarella, Arturo Vidal and Sebastian Giovinco left the Blues facing the prospect of becoming the first holders to depart the competition before Christmas.
Chelsea slumped to third place in Group E with one match remaining and must win against Nordsjaelland in two weeks' time and hope Shakhtar Donetsk beat Juve, despite the Ukrainian side having qualified for the last 16 themselves tonight.
That would be the equal of any let-off the European champions enjoyed en route to last season's astonishing Champions League triumph, which ultimately prevented owner Roman Abramovich dispensing with Di Matteo.
With Pep Guardiola supposedly waiting in the wings, Di Matteo could find himself on borrowed time barring another dramatic turnaround.
The same could be said of the Chelsea career of Fernando Torres, who was axed, although David Luiz produced enough hapless moments for the both of them.
Di Matteo's other changes prompted memories of Andre Villas-Boas's so-called 'suicide-note' selection at Napoli last season, the Blues boss playing without a recognised striker and fielding Cesar Azpilicueta in front of Branislav Ivanovic.
If the line-up was meant to ease the defensive jitters that had seen Chelsea fail to keep a clean sheet for nine matches, it took less than four minutes for them to re-emerge when they twice nearly gifted Juventus the lead, Stephan Lichtsteiner going close before Petr Cech somehow turned his volley onto the post.
The Blues were being penned in but almost scored a sensational breakaway goal in the ninth minute when Oscar ran 70 yards, beating three men before feeding Eden Hazard, whose shot ricocheted behind off Gianluigi Buffon's legs.
Normal service resumed and, when Chelsea fell asleep at a corner, Claudio Marchisio forced another smart Cech stop.
Juve were getting in behind far too easily and Quagliarella twice sent weak shots at Cech, as did Mirko Vucinic on the turn and Andrea Pirlo with a free-kick.
The pressure continued with Hazard mostly isolated, although he was certainly holding the ball up better than Torres had managed of late.
But Oscar almost produced a goal out of nothing again when he raced onto Ramires' pass, Leonardo Bonucci getting back to block.
That proved crucial as Juventus took the lead in unconventional fashion seven minutes from half-time, Quagliarella reacting to redirect Pirlo's low drive past Cech, who could not keep the ball out having already moved to cover the original shot.
It would have been 2-0 seconds later but for the latest incredible goal-line clearance in Ashley Cole's career, the full-back getting back to prevent Kwadwo Asamoah's cross being bundled home by Lichtsteiner.
Chelsea immediately surged upfield but Juan Mata could not prod Hazard's cross past Buffon.
Forced onto the front foot, the visitors ended the first half on top but they were back to leaving far too much space in behind after the restart.
They survived a penalty scare inside five minutes when Vucinic threw himself down under pressure from Gary Cahill and wasted a free-kick of their own when Mata hit the wall.
Pirlo was seeing more and more of the ball and his long-range shot was deflected wide in between Quagliarella sending another tame shot at Cech and failing to round the goalkeeper after Luiz played him onside.
Di Matteo took action by sending on Victor Moses for Azpilicueta on the hour but Chelsea were 2-0 behind a minute later in shambolic circumstances.
Their defence was nowhere to be seen as Asamoah picked out Vidal, whose shot deflected off Ramires and through the admittedly luckless Cech's legs.
Lichtsteiner departed for Martin Caceres but there was no sign of a comeback as Juve threatened a third goal before Di Matteo finally turned to Torres, with John Obi Mikel making way.
Vucinic produced a Torres-like miss six yards out and Ramires was booked for sending Giorgio Chiellini sprawling.
Chelsea tried to respond but Torres made no impression and Giovinco came off the bench to seal the game on the break, poking the ball beyond the onrushing Cech from 20 yards.