Speaking to Bloomberg, General Keith Alexander said that to his knowledge, such acts "never happened". He added that similar allegations levied against the NSA last June were "factually incorrect".
"The servers and everything we do with them, those companies work with us, they are compelled to work with us. It is compelled and these are specific requirements which come from a court order," he said.
An NSA project known as MUSCULAR, revealed by the Washington Post on Wednesday, appears to show diagrams demonstrating how data moving between systems belonging to Google and Yahoo can be tapped by agency equipment.
Alexander rebuffed the claims, saying: "It would be illegal for us to do that. I can tell you factually that we do not have access to Google servers, Yahoo servers. We go through a court order. We issue that court order to them through the FBI."
He added that the extent of the NSA's surveillance had also been exaggerated, claiming it made thousands of requests rather than millions.
Google was said to be "troubled" by the allegations, while Yahoo simply denied any knowledge of such activities taking place. Many tech companies including Microsoft and Facebook had previously been implicated in claims of providing backdoor access to the NSA, which they have repeatedly denied.
Multiple firms have filed petitions with the US government in a bid to be able to publish more transparent information on how they co-operate with the US security services.