Unlike the earlier meeting, which lasted for more than an hour, Gambari met Suu Kyi for only 15 minutes.
But that a second meeting was held at all came as a surprise, raising hopes that Gambari's shuttle diplomacy was making some progress.
The second meeting came after the UN envoy's return from Naypyidaw, Myanmar's new capital, where he held talks with Senior General Than Shwe, the country's ruling general.
Gambari had waited for days to see the reclusive Than Shwe and was expected to express international condemnation after security forces violently put down protests led by Buddhist monks.
"Where are the peace and human rights defenders of the world (the super powers)? They haven't done enough in this case. Isn't there oil in Myanmar?"
Lost Soldier, Arusha, Tanzania
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It appears that last week's military action has already largely succeeded in dispersing the crowds from the streets.
The crackdown is said to have left at least 13 people dead and more than 1,000 arrested.
Dissident groups, though, say up to 200 protesters were slain and 6,000 detained during the government's crackdown last week.
Al Jazeera's special correspondent in Myanmar, who remains annonymous for security reasons, reported on Tuesday: "Any vestiages of resistance from the demonstrators and protesters is probably over now... All the main shrines are open. There are fewer soldiers, significantly, on street corners."
"It seems ike the backbone of the protesters has been broken for now," he said.
Speaking about Gambari's meeting with Than Shwe, he said: "State media has mentioned nothing about it. If you look in the state newspapers, you will see only condemnation of what angry rioters have done to the armed services."
After his meeting with Suu Kyi, Gambari headed to the airport in Yangon, where he was expected to board a flight to Singapore, witnesses said.
The military lightened its presence in Yangon on Tuesday as the city remained quiet after last week's demonstrations.
"Normalcy has now returned in Myanmar," Nyan Win, the country's foreign minister, told the UN General Assembly's ministerial meeting on Monday in a speech defending his country's actions.
He accused "political opportunists" backed by foreign powers of trying to derail its move towards a "disciplined democracy".
He blamed "neo-colonialists" - a reference to the US and other Western nations – for imposing economic sanctions and accused them of spreading disinformation that Myanmar's government was committing human rights violations.
"Recent events make clear that there are elements within and outside the country who wish to derail the ongoing process [towards democracy] so that they can take advantage of the chaos that would follow," he said.
The foreign minister said "political opportunists" exploited a small protest against the rise in fuel prices and "sought to turn the situation into a political showdown aided and abetted by some powerful countries".
He made no mention of the deaths or injuries in the crackdown against the pro-democracy demonstrators.