EU Commission Presidnet Ursula von der Leyen said Wednesday there is "no excuse for hitting a hospital full of civilians" in Gaza, but did not apportion blame for the blast.
Von der Leyen told EU lawmakers the "facts need to be established" on the overnight strike on the Gaza hospital which killed at least 200.
Israel and Palestinians accuse each other of the blast, which triggered street protests in the Middle East against Israel.
Von der Leyen said "all those responsible must be held accountable".
European Council President Charles Michel posted on social media that there was "no conceivable reason to strike a facility with innocent civilians and medical staff" and said such an attack "is not in line with international law".
He too called for accountability.
European Parliament President Roberta Metsola described the hospital attack as "horrific and unjustifiable".
"We must ensure we continue to shed light on what happened," Metsola told parliament. "We must remain clear that protecting civilian lives must keep being a priority. We cannot lose sight of the humanitarian consequences. This has always been our core message."
Von der Leyen and Metsola, who visited Israel last Friday in a show of solidarity, have been criticised for perceived bias in favour of Israel at the expense of Palestinian civilians.
Von der Leyen also faced strident criticism from some EU member states for having met with Israeli Prime Minister Benjamin Netanyahu during that visit and pledging European support - something member states noted she does not have a mandate to do.
The criticism prompted Michel to host a summit of EU leaders by videoconference late Tuesday which emphasised that Israel has the right to defend itself following Hamas's bloody assault -- but only in line with its commitments under international humanitarian law.
The hospital attack and controversy over who was behind it has further fuelled international division. US President Joe Biden said on Wednesday he believed Israel's account of the blast, while several Arab countries, including Israel's allies the UAE and Bahrain, accused it of having bombed the hospital.
Hamas on October 7 sent fighters through the Gaza Strip's heavily militarised border, killing more than 1,400 people. They also took nearly 200 hostages.
Israel has been relentlessly bombing Gaza in response, killing over 3,000 people, according to the health ministry in the Hamas-controlled territory.
In her parliamentary address, von der Leyen said Hamas was the underlying reason for the ordeal Palestinians are now going through.
"Hamas are terrorists. And the Palestinian people are also suffering from that terror," she said.
She added that the EU needs to keep supporting the Palestinians, "and there is no contradiction in standing in solidarity with Israel".
"Europe stands with Israel in this dark moment," von der Leyen said, adding that "Israel should act as a democracy, in line with international humanitarian law."
Against 'rules of war'
The EU's top foreign policy official, Josep Borrell, took a sterner view of Israel's reaction, which has resulted in a siege of Gaza and its 2.3 million Palestinians, who are now cut off from water, food, electricity and humanitarian aid.
"Cutting water supplies and food off from civilian populations isn't in line with the rules of law," Borrell told the European Parliament.
"We cannot make the people of Gaza responsible for the terrible actions of Hamas."
The speeches to the parliament, and Tuesday's videoconference EU summit, highlighted divergences within the EU over the Israel-Hamas conflict.
While there has been widespread condemnation of the slaughter by Hamas of Israelis, disagreements over calls to rein in retaliatory strikes on Gaza continue to boil.