"We have heard voices on both sides of the Atlantic calling for America and Europe to part ways," Stoltenberg said, ahead of wrapping up his decade in charge on October 1.
"Focusing on short-sighted national interests over long-term cooperation will not serve us well. Isolationism will not keep anyone safe."
The warning comes as Washington's allies fret that former president Trump could loosen the commitment of key power the United States to NATO if he wins the election in November.
Trump has rattled European countries by saying the United States could stop protecting NATO members that do not spend enough on defence.
Stoltenberg is set to hand over the reins of the Western military alliance to former Dutch prime minister Mark Rutte next month after a tumultuous 10 years.
During that time, he has helped oversee a major increase in defence spending from European members spurred by pressure from Washington and, more importantly, Russia's war on Ukraine.
But Stoltenberg said that in the face of the ongoing threat from Moscow they will need to up spending further going forward.
At the last count, 23 of NATO's 32 countries were set this year to reach the alliance's target of spending two percent of their gross domestic product on defence, set in 2014.
"The good news is that we have delivered on the pledge we made 10 years ago," Stoltenberg said.
"The bad news is that this is no longer enough to keep us safe."
As Moscow's war on Ukraine grinds on through its third year, Stoltenberg insisted that NATO allies need to ensure Kyiv can negotiate "from a position of strength" when the time comes to talk.
"Any future peace deal must be backed by strong and sustained military support. Not just pieces of paper," he said.
The former Norwegian prime minister conceded that NATO countries "could have done more" to potentially avert Moscow's all-out attack by arming Ukraine earlier.
"If we had been earlier, stronger in our support to Ukraine at least the threshold for Russia to attack Ukraine would have been higher," he said.
- 'More important than ever' -
NATO's ties with Russia have been cut in the wake of the Kremlin's all-out invasion of Ukraine in 2022.
Stoltenberg argued that at some point the alliance needs to return to dialogue with Moscow on issues such as arms control -- but its words must be backed up by power.
"We have to speak to our neighbours. However difficult it might be. But dialogue only works when it is backed by strong defences," he said.
Stoltenberg warned that doing business with rivals such as Russia and China should never come at the expense of security -- and that NATO allies should avoid any reliance on Beijing for key products.
"Freedom is more valuable than free trade," he said.
But he also cautioned NATO members that "protectionism against allies does not protect our security".
France has spearheaded calls for Europe to build up its own defence industry instead of spending on weapons from the United States or Britain.
Trump has also threatened to impose sweeping tariffs on goods coming into the United States.
One of the most painful chapters of Stoltenberg's time in charge was the catastrophic US-led withdrawal from Afghanistan that left the Taliban to take over.
"Military power has its limits," Stoltenberg said.
"We need to be honest about what we can and cannot achieve."
Russia's aggression has reinvigorated NATO -- originally set up to face off against the Soviet Union -- after questions over its purpose following the end of the Cold War.
Since Moscow launched its all-out invasion, the alliance has bolstered its eastern flank, redrawn defence plans and upped spending.
"During my time as secretary general, the relevance of NATO has been questioned. The alliance has been described as divided, obsolete, braindead," Stoltenberg said.
"But the reality is that NATO is strong, united, and more important than ever."
He said his successor's biggest task will be to keep the 32 members of the disparate alliance on the same page.
"That's a big family, a great family, but sometimes what is a challenge, to keep them all happy at the same time," he said.
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