Saudi Arabia, the UAE, Yemen, Bahrain and Egypt all severed diplomatic ties with Doha. Some also announced a cut in transport links.
Gas-rich Doha, which has long exercised an independent streak in its foreign policy, last month denied comments which appeared on its official news agency questioning US hostility towards Iran.
Qatar blamed hackers for a string of explosive remarks attributed to its Emir Sheikh Tamim bin Hamad Al-Thani after Trump visited Saudi Arabia, its giant neighbour.
The reputed remarks were a slap in the face to Riyadh and Abu Dhabi, Sunni monarchies which have embraced Trump and his harder line against Shiite-dominated Iran.
On Monday, Saudi Arabia, the UAE, Yemen, Bahrain and Egypt all severed diplomatic ties with Doha. Some also announced a cut in transport links.
"This certainly represents an unprecedented uptick in tensions within the GCC," the six-nation Gulf Cooperation Council, said Adam Baron, visiting fellow at the European Council on Foreign Relations.
"Qatar has long had an independent streak that's led to resentment from its neighbours," notably Saudi Arabia and the Emirates, and particularly over Qatar's ties with the Muslim Brotherhood, Baron said.
The Islamist group was founded in Egypt about 90 years ago and has spread throughout the region.
Saudi Arabia, Egypt and the UAE all declared it a "terrorist group" after a Brotherhood government, led by Mohamed Morsi, won democratic elections in Egypt before being overthrown by the military in 2013.
Qatar backed Morsi, in a dispute that led Bahrain, Saudi Arabia and the UAE to withdraw their ambassadors from Doha for several months in 2014.
Doha nonetheless has continued to shelter many leaders of Morsi's Brotherhood.
Jane Kinninmont, a senior research fellow at London's Chatham House, said the renewed tensions were not clearly linked to "something new that Qatar has done".
But with Riyadh and Abu Dhabi boosting ties with the Trump administration, the action against Qatar seems like "an attempt to seize an opportunity", Kinninmont said.
In a speech in Riyadh last month, Trump urged Muslim leaders from the Gulf and further afield to "drive out" extremists and "terrorists", as Sunni jihadists carry out attacks in many countries.
But he also named Iran for allegedly fuelling "the fires of sectarian conflict and terror", echoing views long articulated by Saudi Arabia.
James Dorsey, a senior fellow at the S. Rajaratnam School of International Studies in Singapore, pointed Monday to "a Saudi and UAE-driven campaign to isolate Qatar and by extension Iran".
The campaign aims to force non-Arab states to take sides and "persuade the Trump administration to come down hard on Qatar because of its refusal to join the anti-Iranian Saudi bandwagon and its ties to Islamist and militant groups," Dorsey wrote in a briefing paper.
Qatar is also home to the former leader of Hamas, Khaled Meshaal, who has lived in exile for several years in Doha.
In his Riyadh speech against extremism, Trump singled out Hamas, which rules the Gaza Strip, the Islamic State group, Al-Qaeda, and Lebanon's Hezbollah, which is backed by Iran.
Qatar has been criticised for supporting Islamist rebels in Syria, and in 2013, the Afghan Taliban opened a Doha office.
After Trump's call for a clampdown on Islamic extremism, Riyadh's Deputy Crown Prince Mohammed bin Salman and Abu Dhabi Crown Prince Mohammed bin Zayed Al-Nahyan feel empowered "to take ever more radical steps to contain those forces", said Andreas Krieg of the Defence Studies Department at King's College London.
The Saudi prince, who is pushing economic and social reforms at home, admires his Abu Dhabi counterpart for seeing political Islam as a threat to "the Emirati vision of a liberal society emulating the United States".