"We have seen that there has been a significant increase in different attempts to try to interfere by different means in domestic political crises," Stoltenberg told reporters after talks with Spanish Prime Minister Mariano Rajoy.
"We discussed and addressed in our meeting the reports we have seen about attempts by Russia to interfere in several countries," he said.
"Any interference from outside is, of course, unacceptable, and we take this very seriously," he said, adding that NATO has strengthened its cyber-defences.
The Spanish government denounced in November a campaign of "misinformation" during the crisis over Catalan separatists' bid to split from Spain, alleging that messages on social networks came mainly from Russia, but also Venezuela.
- Spain tells EU it suspects Russia meddling in Catalonia
- Russia slams Catalonia claims as groundless
- Catalonia meddling claim has damaged relations with Russia
Madrid has not directly pointed the finger at Moscow, but a report published this month by the Spanish Institute for Defence Studies, a government think-tank, said that the Kremlin "took advantage" of the Catalan crisis to "destabilise" the country following a social media campaign intended to create confusion.
"Moscow seeks to foment discord in Catalonia," according to the report, which warns the "same strategy could be replicated in future in other European states".
In November Moscow rejected the Spanish suspicions as "groundless", likening them to "hysteria" in the United States, where investigations are ongoing into alleged Russian interference in the 2016 presidential election campaign.
Stoltenberg also welcomed Wednesday's announcement by Madrid that it would increase military spending by 73 percent by 2024, even though it will miss the target of 2.0 percent of GDP set by NATO.
"I encourage them to continue in this direction," he said.