Dame Rosemary Butler said: "On balance, the risk to the Siambr (chamber) and potential for disruption to business was too great."
It emerged on Sunday that the official assembly body, the Assembly Commission, had not been consulted on the matter.
Commission member Peter Black AM has said he would have allowed filming.
In a statement on Tuesday, Dame Rosemary noted there had been "much criticism" of her decision, but said: "When I am asked to make a decision I have to weigh up the factors involved in each request.
"In this case, as well as recognising the potential advantages, I also considered the probable scale of what was being proposed and the likely impact on the Siambr given the number of people and amount of equipment involved in a production of this type, and the adjustments to the Siambr which would have been involved.
"We offered other areas of the Senedd but this was not taken up."
She added: "I am disappointed that the discussion has portrayed the Assembly as a body that is not open to do business with the creative industries", saying programmes such as Doctor Who and Sherlock had been filmed on assembly property.
Meanwhile Welsh Secretary Stephen Crabb said the decision to refuse permission to film in the Senedd chamber was "quite bizarre".
He told MPs that the opportunity of shooting scenes in Cardiff Bay featuring "possibly the best-known film character" the movie industry had produced seemed "to have been lost".
The decision to reject a request to film scenes for a James Bond movie in the Senedd chamber has been defended by the assembly's presiding officer.