The big guns from Group B return to action after Russia all but guaranteed their place in the next round, sweeping Egypt aside 3-1 to make it two wins out of two.
Five-time world player of the year Ronaldo bagged a sublime treble to salvage a 3-3 draw against Spain in Sochi in Russia 2018's stand-out match so far, the 51st of his glittering career.
"I'm very happy, it's a nice personal record to have, one more in my career," Ronaldo said.
"For me the most important thing is to emphasise what the team did against one of the favourites to win this World Cup."
Spain head to Kazan to face Iran still counting the cost of a chaotic run-up to the tournament during which Julen Lopetegui was sensationally sacked just two days before they opened their campaign.
Former international defender Fernando Hierro is now at the helm and has the job of steadying the ship and steering Andres Iniesta and his teammates into the last 16.
"It's a crucial match that will determine our World Cup destiny," said midfielder Isco of the Iran showdown.
"We want to score from the opening minute, even though that won't be easy."
Iran kicked off their campaign with a 1-0 win against Morocco, thanks to a late own goal by Aziz Bouhaddouz, piling the pressure on Spain at Kazan Arena on Wednesday.
Iran sit on top of the pile as the only Group B team with a win so far, an achievement that sparked celebrations in the streets of Tehran and throughout the Islamic republic.
Coach Carlos Queiroz concedes Iran now face one of the biggest challenges in their World Cup history, but maintains they can "make the impossible possible".
"We are well aware of Spain's reputation, so we'll have to play our best game ever. In the end the football gods will decide," said the former Real Madrid coach.
Iran have never beaten a European side at a World Cup -- their only previous victory was a 2-1 win over the United States in France in 1998.
Morocco need to bounce back from their agonising defeat against Iran but Portugal, and Ronaldo in particular, pose Herve Renard's men a monumental challenge at Moscow's Luzhniki Stadium.
"We lost a battle but we haven't lost the war. We're not dead," said midfielder Faycal Fajr, one of 17 foreign-born players in Morocco's squad.
100 up for Suarez
Uruguay take on Saudi Arabia in Wednesday's other game, with forward Luis Suarez set to mark his 100th appearance for the Celeste in the Group A clash in Rostov-on-Don.
The Barcelona man has exited the last two World Cups in disgrace after being sent off for a goal-line handball against Ghana, and he infamously sank his teeth into Italy defender Giorgio Chiellini in 2014.
The game itself should be straightforward for Uruguay, who won their Group A opener 1-0 against an Egypt team without Mohamed Salah as they face a Saudi Arabia outfit hammered 5-0 by hosts Russia in the tournament curtain-raiser.
The Saudis' preparations were jolted by a mid-air mishap on Monday when the plane carrying them to Rostov suffered a fire in one of its engines.
The aircraft landed safely after what one of the Saudi players called a "simple malfunction".
Hosts Russia stand on the verge of the knockout stages after Tuesday's emphatic win against Egypt in Saint Petersburg.
After a goalless first half, Egypt captain Ahmed Fathi scored an own goal before Denis Cheryshev struck and Artem Dzyuba added a third. Salah scored a consolation penalty late on but it was not enough for the Pharaohs.
A grinning Prime Minister Dmitry Medvedev posted a photo on Facebook of him watching the Egypt game with his Belarussian counterpart Andrei Kobyakov.
"Victory!" he wrote.
Earlier, Senegal secured a first win for an African nation at this tournament, benefiting from a terrible error from Polish goalkeeper Wojciech Szczesny to beat Poland 2-1 in Moscow.