The goal was enough to clinch a 4-3 aggregate win for Madrid, as the two-time defending champion reached the semifinals for the eighth straight season.
And Madrid captain for the night, Marcelo, took the opportunity to take a pop at Spanish rivals Barcelona after his side's victory.
But Ronaldo was the coolest man in a boiling Bernabeu, and he hammered the spot kick into the top corner - keeping his run of scoring in all 10 Champions League games this season going - and sending the holders through to the last four.
Zinedine Zidane was forced to rethink his gameplan in tonight's Champions League quarter final return leg after his side found themselves two goals down to Juventus in the first 45 minutes - and with the Italians requiring just one goal to level the tie.
The manner of the loss, with Juve fighting back, coming so close to extra time and losing to a late Ronaldo penalty has triggered an emotional reaction among those associated with Madrid and Juventus.
After Ronaldo drew a comfortable save out of Buffon on 57 minutes after being picked out by substitute Lucas Vazquez, Higuain tested Navas two minutes later following good work by Matuidi and Miralem Pjanic to set him up on the edge of the box. "That allowed their team to grow and it made things hard for us". The crowd got nervous when Mario Mandzukic added his second goal before halftime, and the unlikely comeback was almost complete when Blaise Matuidi scored the third about 15 minutes into the second half.
"Real Madrid could have scored more goals today, but Juventus and Buffon were great".
Mario Mandzukic's first goal after 76 seconds was the fastest that Real Madrid have ever conceded in a Champions League home game.
"It is obvious that Real Madrid always benefit in the Champions League and that it never goes the other way".
"There are no favorites in this stage", Madrid right back Dani Carvajal said.
When Real's penalty was given in stoppage time, the match was delayed for a few minutes as tempers frayed, during which time Ramos was shown in heated conversation with Juve coach Massimiliano Allegri.