Ronaldo became the first player to feature in six European Championships after being selected in Roberto Martinez's starting lineup.
The 39-year-old had opportunities to mark the historic occasion with a goal, going close on three occasions during the first period, heading wide in the early stages before drawing a couple of strong stops from Jindrich Stanek.
After dominating the first hour, Portugal were left stunned when Lukas Provod produced a stunning strike to give the Czech Republic a surprise lead.
Unfortunately for Ivan Hasek's side, their slender advantage lasted just seven minutes before they conceded an equaliser as Stanek pushed the ball onto Robin Hranac, who could only watch it roll into the net.
Portugal thought they had been denied the chance to claim all three points after Liverpool's Diogo Jota had a goal ruled out, but that was all forgotten when Conceicao slotted home a dramatic 92nd-minute winner.
Ronaldo fails to convert first-half chances
Despite making history with his mere presence on the rain-drenched pitch, Ronaldo was hungry to offer a meaningful contribution and add to his staggering 130-goal international tally.
Ronaldo got his first sight of goal in the eighth minute, sending the ball wide after rising high to meet Rafael Leao's lofted cross.
With an array of world-class talent, it was unsurprising to see Portugal dominate possession, challenging the Czech Republic to remain disciplined and compact in their defensive shape.
As the contest moved past the half-hour, Manchester United's Bruno Fernandes carved open a stubborn Czech Republic backline, producing a sublime pass to send Ronaldo through on goal, but the Al-Nassr forward could find no way past the sprawling Stanek.
Ronaldo then threatened on the stroke of half time, chopping onto his left foot before stinging Stanek's palms with a powerful effort.
Provod's strike proves to be fruitless
After failing to break the deadlock before the break, the Selecao continued to probe and push for the all-important opener in the second period.
However, when the opening goal finally arrived it was the Czech faithful who were celebrating a sensational long-range shot.
Provod latched onto Vladimir Coufal's neat layoff to whip an unstoppable strike into the far corner, sparking scenes of jubilation among the Czech contingent.
Their lead ultimately proved to be brief as Stanek failed to adequately deal with Nuno Mendes's downward header, pushing the ball against the unfortunate Hranac, causing it to roll into the awaiting net.
Jota then thought he had netted an 87th-minute winner, tucking the ball home after Ronaldo crashed his header against the upright.
However, a swift VAR review revealed that the former Real Madrid forward had just strayed offside, offering a reprieve to the exhausted Czech Republic side.
But, just as the Czech Republic started to believe they had done enough to clinch a point, Conceicao came off the bench to make a decisive intervention in his third international appearance.
The Porto midfield took advantage of Hranac's failure to deal with a Pedro Neto cross, firing the ball home from close range to snatch all three points in the most dramatic of fashion.
What next for Portugal and Czech Republic?
After celebrating an impressive turnaround, Portugal will focus on Saturday's tricky encounter against a Turkey side that will be full of confidence following their 3-1 win over Georgia.
Meanwhile, the Czech Republic will have to dust themselves off in time for their second group game against Georgia at the Volksparkstadion in Hamburg.