The White House revealed details of the telephone call between the two leaders, saying the US President expects a path towards a ceasefire to develop.
The call comes after 10 days of heavy fighting between Israelis and Palestinians.
In his toughest message to Israel since the conflict erupted, the president asked Mr Netanyahu to move towards "the path to a cease-fire," according to a White House statement on their conversation.
However, Mr Netanyahu vowed to press ahead with a fierce military offensive in the Gaza Strip.
Netanyahu's comments marked the first public rift between the two close allies since the fighting began last week and could complicate international efforts to reach a cease-fire.
After a visit to military headquarters, Netanyahu said he “greatly appreciates the support of the American president”, but said Israel will push ahead “to return the calm and security to you, citizens of Israel”.
He said he is "determined to continue this operation until its aim is met."
Pressure has also been mounting Mr Biden to do more as the death toll in the conflict has topped 200.
"The two leaders had a detailed discussion on the state of events in Gaza, Israel's progress in degrading the capabilities of Hamas and other terrorist elements, and ongoing diplomatic efforts by regional governments and the United States," White House spokeswoman Karine Jean-Pierre told reporters.
"The president conveyed to the prime minister that he expected a significant de-escalation today on the path to a ceasefire," she added.
In the latest clashes on Wednesday, Israeli airstrikes killed at least six people across the Gaza strip and destroyed the home of an extended family.
The military said it widened its strikes in the Palestinian territory's south to blunt continuing rocket fire from Hamas, while a separate barrage also came from Lebanon.
Residents surveyed the piles of bricks, concrete and other debris that had once been the home of 40 members of al-Astal family. They said a warning missile struck the building in the town of Khan Younis five minutes before the airstrike, allowing everyone to escape.
Ahmed al-Astal, a university professor, described a scene of panic before the airstrike hit, with men, women and children racing out of the building. Some of the women didn't even have time to cover their hair with Muslim headscarves, he said.
"We had just gotten down to the street, breathless, when the devastating bombardment came," he said. "They left nothing but destruction, the children's cries filling the street. ... This is happening, and there is no one to help us. We ask God to help us."
The Israeli military said it struck a militant tunnel network around the towns of Khan Younis and Rafah, with 52 aircraft hitting 40 underground targets. Gaza's Health Ministry said a woman was killed and eight people were wounded in those strikes.
The latest strikes came as diplomatic efforts aimed at a cease-fire gathered strength and Gaza's infrastructure, already weakened by a 14-year blockade, rapidly deteriorated.
Medical supplies, water and fuel for electricity are running low in territory, on which Israel and Egypt imposed the blockade after the Islamic militant group Hamas seized power in 2007.
At least 219 Palestinians have been killed in the current fighting, including 63 children and 36 women, with 1,530 people wounded, according to the Gaza Health Ministry.
Hamas and Islamic Jihad say at least 20 of their fighters have been killed, while Israel says the number is at least 130. Some 58,000 Palestinians have fled their homes.
Twelve people in Israel, including a five-year-old boy and a soldier, have been killed.