Iran's insistence its nuclear programme was peaceful could not be confirmed, Yukiya Amano told the International Atomic Energy Agency (IAEA) in Vienna.
Mr Amano is taking a much more critical line than the organisation has done before, correspondents say.
The news is likely to strengthen calls for sanctions on Iran.
"The agency continues to verify the non-diversion of declared nuclear material in Iran, but we cannot confirm that all nuclear material in Iran is in peaceful activities because Iran has not provided the agency with the necessary co-operation," Mr Amano said.
He was speaking at a 35-nation board meeting of the IAEA in Vienna, the first since he took over as the head of the organisation in December.
Sanctions call
The BBC's Bethany Bell in Vienna says Mr Amano's address, and a report leaked earlier this month, are much more critical of Iran than the IAEA had been under its former head, Mohamed ElBaradei.
This could strengthen calls for tough sanctions on Iran, which have so far been resisted by UN Security Council members Russia and China, our correspondent says.
Iran insists its nuclear enrichment programme is for peaceful civilian use. It says it wants to increase the enrichment of uranium it has for power generation and for medical use.
It accuses the IAEA of being influenced by the US.
But critics say that Iran's attempts to enrich uranium to 20% are the first step to creating weapons-grade nuclear material.
Last year, Iran revealed a previously unknown nuclear facility in the mountains near the city of Qom.
Tehran has said it is planning 10 further sites to enrich uranium.
