Secretary-General Jens Stoltenberg said Russia had not provided "any real explanation" of the violation, which "lasted for a long time."
Russia says Saturday's incursion was brief and due to bad weather. It is examining claims of another violation.
Turkey's army also says an unidentified fighter jet locked its radar on to eight of its jets on Monday.
It echoes a similar incident on Sunday, when an unidentified Mig-29 - which analysts say may have been Syrian - locked its radar onto Turkish jets for more than five minutes over the Turkish-Syrian border.
Missile systems inside Syria were also locked on to Turkish planes for more than four minutes on Monday, the Turkish military says.
The incidents involving Mig-29 aircraft "illuminating" Turkish F-16 jets with their radars - a preliminary to actually engaging them - suggests a new assertiveness on the part of the Syrian air force.
Russia, as far as we know, has not deployed Mig-29s as part of its air expeditionary force to Syria. It has though supplied its Syrian counterpart with the aircraft in the past.
Some - at least - of Syria's Mig-29s are still operational; indeed, as a fighter rather than a ground attack aircraft, they have flown a lot less during Syria's protracted civil war.
Syria and Turkey have a difficult history of incidents over recent years. In 2012, Syrian missiles shot down a Turkish Phantom jet off the Mediterranean coast.
Last year, Turkish jets shot down a Syrian Mig-23 that had strayed into Turkish airspace along with a Syrian helicopter earlier this year. So the tensions are real and the possibility of a deadly encounter ever present.
Syria still maintains reasonably sophisticated surface-to-air missile defences, but many bases have been overrun and it is far from an integrated national system.
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On Tuesday, Syrian state TV said Russia had hit parts of Palmyra, which is held by Islamic State (IS) militants and is renowned for its nearby ancient ruins, but Russia denied the claims.
The UK-based Syrian Observatory for Human Rights said 15 IS fighters were killed in the Palmyra air strikes.
The Russian defence ministry said it flew 20 sorties on Tuesday, striking 12 IS targets.
It said field camps, explosive stores and food supplies were destroyed in the north-western province of Idlib, where rebel groups have made significant gains against government forces in recent months.
Russia began its air campaign in Syria last Wednesday.
It says it is targeting "all terrorists" in co-ordination with Syria's government, but Nato and allied states have expressed concern that it is concentrating its attacks on rebel groups opposed to President Bashar al-Assad, some of them backed by the West, and not jihadist groups like IS.
Turkey has twice summoned the Russian ambassador - once over the first violation, which occurred on Saturday, and once over a second violation that Turkey says took place on Sunday.
In the latest reaction:
Mr Stoltenberg called the Russian violation "unacceptable", saying Nato was taking it "very seriously" and warning that "incidents, accidents, may create dangerous situations"
There had been "a substantial military build-up" by Russia in Syria, including ground troops and naval capabilities, he said.
Russian President Vladimir Putin has denied that civilians have been killed by Russian strikes in the past week, but evidence on the ground has indicated otherwise.
A US-led coalition has been conducting air strikes against IS in Syria and Iraq since September last year, which rights groups say have also caused civilian deaths.
Syria's conflict, which began in 2011, has left more than 250,000 dead and about half the country's population displaced.
Russia's violation of Turkish airspace over the weekend "does not look like an accident", Nato has said.