Iran's opposition faces a critical test of resolve and the country an uncertain future tomorrow after the Islamic regime's supreme leader, Ayatollah Ali Khamenei, issued a blunt warning to those involved in mass protests over last week's "stolen" presidential election that they would "bear the responsibility" for any bloodshed.

Khamenei rejected accusations of fraud in the poll, confirmed the incumbent, Mahmoud Ahmadinejad, as the winner, and gave no ground to the millions of ­Iranians demanding their votes back.

Mir Hossein Mousavi, who claims he beat Ahmadinejad in the race, was said by an ally to have no plans for unauthorised rallies tomorrow following the warning, but supporters vowed to go on protesting.

Fears grew tonight of an intensifying crackdown on media and opposition activists. Students at the fine arts faculty of Tehran University – where scores of students were injured and some reported killed after raids by security forces earlier this week – announced an indefinite sit-in starting tomorrow.

Khamenei's closely watched speech at prayers at Tehran University could hardly have been tougher. It had been hoped he might adopt a more conciliatory tone that would help defuse the gathering crisis, the worst in Iran's 30-year post-revolutionary history. But he warned: "If there is any bloodshed, the leaders of the protests will be held directly responsible. The result of the election comes from the ballot box, not from the street. Today the Iranian nation needs calm."

Tens of thousands of worshippers cheered as he told them: "It is your victory. They cannot manipulate it."

Mousavi, a moderate former prime minister whose "green" movement scared the regime with the support it was attracting, ignored a call to attend the prayer meeting and now faces a dilemma over his next step. Ignoring Khamenei's message risks bloodshed on a far larger scale than the eight people killed last week. Accepting it means surrender to the regime.

The reformist cleric Mehdi Karroubi, another candidate for the presidency, added to the pressure tonight by also calling for the election to be annulled. "Accept the Iranian nation's will by cancelling the vote and guarantee the establishment's survival," he urged.

Khamenei attacked opponents at home but also lambasted Iran's enemies abroad in hardline remarks that bode ill for any opening to the US, where Barack Obama is seeking talks to tackle worries over Iran's nuclear ambitions.

Britain was attacked as "the most evil", but the US, Israel and "Zionist-controlled" media were also abused, as was Hillary Clinton, the US secretary of state. "The enemies are targeting the Islamic establishment's legitimacy by questioning the election and its authenticity before and after [the vote]," said Khamenei.

President Barack Obama toughened his rhetoric yesterday in support of the demonstrators and criticised the Tehran government for its violent response.

"I'm very concerned – based on some of the tenor and tone of the statements that have been made – that the government of Iran recognise that the world is watching," Obama told CBS News. "And how they approach and deal with people who are, through peaceful means, trying to be heard will, I think, send a pretty clear signal to the international community about what Iran is – and is not."

Analysts and commentators were dismayed by the implications of Khamenei's speech. Sadegh Saba, chief analyst for BBC Persian TV, said: "Mousavi wants the protests to continue but Khamenei is saying if they do there might be bloodshed – and it will be on your hands."

Issa Saharkhiz, a Tehran-based pro-reformist commentator, said Khamenei's speech had transformed the crisis from a conflict over the election result into a trial of his own political authority, which was now being openly questioned. "Now the issue is that the supreme leader's sense of justice, management and competence is under question," he told Deutsche Welle. "The leadership cannot be left in the hands of such a person, who for the sake of preserving himself and his power threatens people with mass murder."

Crucially, Khamenei ruled out any cheating in the election, apparently dashing hopes that a partial recount ordered by the guardian council, a supervisory body of senior clerics, will mitigate the crisis.

Khamenei's call for Mousavi and Karroubi to confine their protests to legal avenues prompted mockery. "This means that Imam Hossein [the third most revered figure in Shia Islam], instead of making a last stand at Karbala, [should have] pursued his grievances through the legal process," one blogger said on the Farsi blogsite Balatarin.

Balatarin was flooded with messages voicing outrage at Khamenei's warning. One read: "Mr Khamenei, the direct responsibility for any damage to people's lives or property now lies with you."