"Any attempt to intervene in, to influence national elections from outside is unacceptable," Stoltenberg said when asked about allegations of Russian cyber attacks and hacking during the 2016 US presidential campaign.
Trump has consistently berated US intelligence agencies over their claim Russian President Vladimir Putin personally approved a cyber offensive to help him defeat Hillary Clinton.
On Wednesday, the president-elect, who took a much softer line on Russia than Clinton, conceded Moscow may have meddled but he bluntly rejected any suggestion his aides colluded with the Kremlin to win the vote.
Russia has rejected the charges.
Stoltenberg insisted the danger was real, saying that the US-led military alliance was "concerned" over cyber security and had seen reports about such attacks against "many NATO allies."
"That is why NATO has focused on cyber defence... cyber security is top of our agenda," he said after a meeting with New Zealand Prime Minister Bill English at NATO HQ in Brussels.
He recalled that NATO leaders at their Warsaw summit in July had made cyber security an "operational domain," putting it on a par with its responsibility to provide traditional military capabilities on land, sea and in the air.
They also endorsed plans for a special response unit to help any ally under threat from a cyber attack against either military or civilian infrastructure.
NATO "does not see any imminent threat against an ally" from Russia, Stoltenberg said when asked about the current threat assessment.
But he charged that a "more assertive Russia" was engaged in a major military build-up and had shown it was ready to use force to get its way, as demonstrated in Georgia and Ukraine.
Asked about Trump's commitment to NATO -- which the President-elect appeared to undercut during the campaign -- Stoltenberg said he had no doubts.
"I am absolutely certain the new president will stay committed to NATO, to (US) security guarantees. That was the clear message when I spoke to Donald Trump some days after the election," he said.