The United States has frequently accused Iran of providing some assistance to insurgents in Afghanistan, although Washington says it has not been nearly as important a factor as in Iraq, Iran's other neighbor where U.S. troops are waging war.
"Iran is working to increase its influence in the area. On the one hand, that's not surprising, she is a neighbor state, a neighbor country. On the other hand, the influence I see is all too often negative," Mullen told a news conference during a visit to Kabul, in response to a question about Tehran's influence.
"I was advised last night about a significant shipment of weapons from Iran into Kandahar, for example," Mullen said.
"I have seen them over the last several years — the last couple of years anyway, certainly be more than just interested, provide some capabilities," Mullen added. "I am also concerned that that desire to be influential is increasing."
Pentagon officials declined to give further details about the Iranian arms shipment. Asked later if it represented an important development, Mullen said: "I was taken aback. It wasn't insignificant."
Tehran denies supporting militant groups opposed to President Hamid Karzai's government, and blames the presence of Western troops in Afghanistan for causing instability.
Mainly Shi'ite Muslim Iran was strongly opposed to the strict Sunni Muslim Taliban when they ruled Afghanistan from 1996-2001.
Iran has had rapidly growing economic influence in Afghanistan, especially in the West, where cross-border trade is brisk. A dialect of Iran's Farsi language is one of two state languages in Afghanistan, and Iran hosted millions of Afghan refugees during decades of war.