“The Iranian regime openly advocates another Holocaust and it seeks the means to achieve it,” Mr. Pence said, in a speech delivered in a country where occupying Nazi forces housed the Auschwitz extermination camp during World War II.

Mr. Pence’s message came as a surprise to European countries that had been reassured by U.S. officials that the two-day conference on peace in the Middle East wasn’t solely an effort to build a coalition against Iran, a Western diplomat said.

“At the outset of this historic conference, leaders from across the region agreed that the greatest threat to peace and security in the Middle East is the Islamic Republic of Iran,” Mr. Pence said. “Countries across the globe are working to cut Iranian oil imports this year.”

“Sadly, some of our leading European partners have not been nearly as cooperative,” he said.

The vice president took particular aim at France, Germany and the U.K., which together defied threats from Washington and in late January set up a special-payments company to in effect circumvent U.S. sanctions on Iran and open an avenue for trade between Europe and Iran.

“They call this scheme a special purpose vehicle; we call it an effort to break American sanctions against Iran’s murderous revolutionary regime,” Mr. Pence said. “It’s an ill-advised step that will only strengthen Iran, weaken the EU and create still more distance between EU and the United States.”

After the conference, the German Foreign Office said in a Twitter post: “EU is a cornerstone of the multilateral system. Compromise and consensus are deeply embedded in its DNA. We are a reliable partner for those who want to uphold the rules-based order and who are prepared to shoulder more responsibility to this end.”

A spokesperson for the U.K. government said: “While we share U.S. concerns about Iranian regional activities and its missile program, we believe the best way to address these wider concerns is while the nuclear deal remains in place.”

France didn’t immediately respond to a request for comment.

Major European countries sent low-level delegations to the U.S.-led Warsaw gathering of more than 60 countries to protest its focus on Iran.

In closed-door sessions at the conference, Iran was repeatedly identified, particularly by Middle Eastern delegates, as the region’s main problem, both in Syria and in Yemen.

Yemen’s Foreign Minister Khaled al-Yemani blamed Iran, which supports the Houthi rebels who captured the country’s capital in 2014, for stoking Yemen’s nearly five-year war. Mr. Yemani, sitting between U.S. Secretary of State Mike Pompeo and Israeli Prime Minister Benjamin Netanyahu, warned the Europeans that Iran’s influence in Yemen could harm them.

“They are using Yemen as a testing ground. They will rain Europe with rockets and missiles whenever they decide to do that,” he said.

Adel al-Jubeir, Minister of State for Foreign Affairs for Saudi Arabia, which supports Yemen’s internationally recognized government, complained that there seemed to be a “moral equivalency” among the international community watching Yemen.

“We didn’t start this. The Houthis did, with the support of the Iranians,” Mr. Jubeir said. “The Houthis are the problem and we need to keep our eye on that problem.”

On Syria, the participants expressed similar concerns about Iran’s efforts to establish a presence there.

Mr. Netanyahu accused Iran of trying to colonize Syria and use it as a base to threaten Israel. “There should be a common international position to say to Iran, ‘get out of Syria.’ It’s actually in Assad’s interest. I think it’s in the interest of just about everyone,” he said in a closed-door session, according to a source present at the meeting.

Mr. Pompeo insisted the conference had covered a broad theme of topics and views in closing remarks, and said the U.S. accepts that other countries don’t share Washington’s viewpoint on Iran.

While Iran wasn’t invited to the Warsaw conference, its president, Hassan Rouhani, met Thursday with two other powers with influence in the Middle East—Russia and Turkey—that had been invited but chose to skip it.

The presidents of the three countries met in Sochi on Russia’s Black Sea coast to discuss the future of Syria.

“While today in Sochi we take a new step together with Russia in order to strengthen stability in the region and fight against terrorism in Syria, some who are supporters of terrorism themselves are plotting in Warsaw against the region,” Mr. Rouhani told Turkey’s Recep Tayyip Erdogan, according to Iranian state media.

—Aresu Eqbali in Tehran contributed to this article.

Write to Jessica Donati at jessica.donati@wsj.com and Sune Engel Rasmussen at sune.rasmussen@wsj.com