Chelsea thought they had handed Mourinho a first trophy of his second spell as boss when Eden Hazard netted in extra time after Franck Ribery had cancelled out Fernando Torres' opener at the Stadion Eden.
The dismissal of Ramires for a second bookable offence left the Blues exposed and Javi Martinez tucked in with the final kick of an astonishing game to make it 2-2, forcing penalties.
The 2012 European Cup final between the sides had been decided on spot-kicks in Chelsea's favour and on this occasion, shooting towards the boisterous Bayern end, substitute Romelu Lukaku saw his effort saved as the Germans claimed a 5-4 shootout success.
It denied Mourinho a first European Super Cup and a maiden trophy of his second spell at Stamford Bridge.
There had also been personal pride at stake for former Inter Milan and Real Madrid boss Mourinho, who had won three of his previous 15 encounters with Guardiola's Barcelona.
Guardiola remains his nemesis.
Bayern, treble winners last season and labelled the "perfect" team by Mourinho on the eve of the match, were without influential midfielder Bastian Schweinsteiger due to an ankle injury, but were dominant in an enthralling encounter.
The Blues were on the back foot for most of the evening, but went in front courtesy of a goal on the break.
Restored to start following a striker-less line-up at Manchester United, Torres was at his clinical best in converting Andre Schurrle's cross, showing Mourinho he is prepared to fight new recruit Samuel Eto'o for his place.
Bayern had been torn apart, but they continued to press forward.
Newly crowned European Player of the Year Ribery was Bayern's most threatening player and Chelsea failed to heed the frequent first-half warnings.
Two minutes after the restart the France forward drifted in off the left and fired in an unstoppable 25-yard shot to equalise.
After Ramires' sending-off, Chelsea regrouped and Hazard replicated Ribery's strike, only from closer in, and Petr Cech made a series of fine saves.
Cech could not deny Martinez, though, as the destination of the trophy was determined by spot-kicks.
With nine successful penalties it came down to Lukaku, but his spot-kick was soft and Manuel Neuer made a comfortable save.
Neither Mourinho nor Guardiola were in charge of their respective teams when they won the competitions which brought them to Prague.
Guardiola, following a year's sabbatical after leaving Barcelona, replaced the now retired Jupp Heynckes and Mourinho succeeded Rafael Benitez, who as interim boss led Chelsea to the Europa League title in a campaign which began in Prague.
The Blues were seeking to make amends for their appearance in the Super Cup last season, when they were beaten 4-1 by Atletico Madrid in Monaco and made two changes from Old Trafford, with David Luiz making his first appearance of the season following a hamstring injury alongside Torres, but again Juan Mata was on the bench.
Bayern fielded nine of the 11 players who began May's European Cup final defeat of Borussia Dortmund at Wembley, but went behind to a Chelsea counter attack, something Guardiola had been wary of.
Hazard surged forward, found Schurrle down the right and he centred the ball for Torres, who struck a first-time shot beyond Manuel Neuer.
Bayern dominated again after the goal, with Chelsea starved of possession and squeezed into their own half, but they had to wait until the second period to level when Ribery succeeded where he had narrowly failed in the first period.
The Frenchman cut in from the left before scoring with a fierce right-foot shot.
Neuer spread himself well to save from Oscar and Frank Lampard fired the rebound over.
Luiz antagonised Bayern at every set-piece and was booked for a foul on Mandzukic in an increasingly feisty encounter.
Toni Kroos and Jerome Boateng had efforts before Neuer acrobatically palmed away a Branislav Ivanovic header and held a Schurrle shot.
Ramires was sent off for a foul on Mario Gotze which earned him his second yellow card, putting Chelsea under further pressure.
Bayern tried to win the game in the remaining five minutes of normal time and Ribery fired wide after a one-two on the edge of the area with Gotze before the whistle blew and Mourinho and Chelsea players hounded the officials.
It was the 10 men of Chelsea who re-took the lead, when Hazard danced in from the left, by the static Martinez and Boateng, before drilling low into the net.
Cech saved a Xherdan Shaqiri effort as Bayern attempted to respond for a second time, but the Blues reached the extra-time interval, knowing they had to survive for 15 minutes more.
Mandzukic was twice denied by Cech and Shaqiri was unable to divert a Martinez header down goalwards.
Mourinho threw Terry into the action, in place of Hazard, for the final seven minutes, as Bayern continued to go forward.
Cech saved from Gotze and Ribery but could not hold out as Martinez netted with the last kick of extra-time to force penalties.
On this occasion, Bayern triumphed.