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Wednesday's attack, which targeted a busload of Revolutionary Guards in the volatile southeastern province of Sistan-Baluchistan, was one of the deadliest on Iranian security forces in years.
The appointment of Kambiz Mehdizadeh, in his early thirties and reportedly married to Rouhani's daughter in a low-key wedding this August, was widely criticised by Iranians on social media.
Tensions have risen between Iran and the United States after US President Donald Trump withdrew from a multilateral nuclear deal in May and reimposed sanctions on the Islamic Republic.
He made the remarks at the 32nd International Islamic Unity Conference which kicked off in the capital Tehran earlier in the day.
Rouhani said the United States cultivates close ties with regional Muslim nations to protect Israel, an apparent reference to Iran's regional rival Saudi Arabia and the kingdom's Sunni Arab allies.
Khashoggi, a government critic who was living in self-imposed exile in the United States, was murdered inside the Saudi consulate in Istanbul on October 2 as he organised the paperwork for his marriag
The US is due to complete the reimposition of sanctions on November 5, targeting Iran's oil sector and central bank.
The United States has moved to reimpose sanctions that had been lifted under the landmark deal to curb Iran's nuclear program and has vowed to punish foreign firms that do business with Iran.
"I am starting the dialogue right here, and state, in unequivocal terms, that the question of international security is not a toy in American domestic politics."
In the immediate aftermath of the attack on Saturday, Iranian authorities said they suspected Arab separatist groups were behind the attack, none of whom is known to have a presence in Syria.
Hassan Rouhani's comments came as Iran's Foreign Ministry also summoned Western diplomats over them allegedly providing havens for the Arab separatists who claimed Saturday's attacks
The Iran summoned diplomats from Denmark, the Netherlands and Britain over their "hosting of some members of the terrorist group" which carried out the attack.
Iran has ballistic missiles with a range of up to 3,500 kilometres (2,200 miles), enough to reach both Israel and US bases in the Middle East.
Their delicate, and perhaps unrealistic, task is to build a case for Tehran to respect the deal's limits on its nuclear program even though Washington has pulled out.
Donald Trump has signalled that he might be willing to meet with Rouhani as well, after re-imposing sanctions against Iran last month.