At 9:49 a.m., the shooter posted a final message on the social media platform Gab. Five minutes later, he opened fire on the synagogue's windows and entered the building armed with a Colt AR-15 rifle and three Glock pistols. Rabbi Jeffrey Myers, upstairs in the Pervin Chapel, initially mistook the first shots for a falling coat rack. Downstairs, Jerry Rabinowitz and Daniel Leger, both with medical backgrounds, ran toward the sounds to help. Rabinowitz was killed; Leger was shot in the abdomen and lay bleeding on the stairs for 45 minutes before rescue. The gunman moved through the building methodically, room by room, basement to chapel, for approximately 20 minutes. Eleven worshippers were killed. The victims ranged in age from 54 to 97.

The dead included two brothers, Cecil and David Rosenthal, who lived together at a community home for people with disabilities. Bernice and Sylvan Simon, a retired couple who had celebrated their wedding at Tree of Life in 1956, died together. Rose Mallinger, 97, had been an active congregant for more than 60 years. Melvin Wax, 87, a retired accountant, was leading New Light's services when the attack began. He hid in a closet with other congregants but opened the door, unaware of what was happening, and was shot at point-blank range. Irving Younger, 69, served as a greeter at the synagogue. Joyce Fienberg, Richard Gottfried, Jerry Rabinowitz, and Daniel Stein rounded out the eleven. Seven others were injured, including four police officers who responded to the scene.

That evening, over 3,000 people gathered at the intersection of Murray and Forbes avenues for an interfaith candlelight vigil organized by students from nearby Taylor Allderdice High School. Rabbis, imams, pastors, and civic leaders spoke. Members of the Pittsburgh Steelers attended the Rosenthal brothers' funeral. The Pittsburgh Penguins wore "Stronger Than Hate" patches and auctioned the jerseys for the synagogue. The University of Pittsburgh altered its Victory Lights atop the Cathedral of Learning to shine for 11 seconds, one for each victim. Pittsburgh locals crocheted Stars of David and hung them along Murray Avenue. The Eiffel Tower darkened. Tel Aviv lit its city hall in the colors of the American flag. Pope Francis prayed for the victims from St. Peter's Square.

The shooter was charged with 63 federal crimes and 36 state counts. His defense offered a guilty plea in exchange for life without parole, but federal prosecutors pursued the death penalty. The trial began on May 30, 2023, after years of pretrial proceedings. Prosecutors called sixty witnesses; the defense called none. Survivors testified, including congregants and Rabbi Myers. After 11 days of testimony, the jury found the defendant guilty on all counts. The sentencing phase weighed his history of radicalization against the planned nature of the attack. On August 2, 2023, the jury unanimously sentenced him to death, the only federal death sentence imposed during President Biden's administration. In December 2024, when Biden commuted 37 of 40 federal death sentences, this was one of three he excluded.

The aftermath reshaped Jewish security in America. The Jewish Federations of North America raised $62 million to secure Jewish communities across the continent. Federal grants through the Nonprofit Security Grant Program brought in $100 million. By 2023, 93 Jewish federations had full-time security directors, a more than fourfold increase in five years. Pennsylvania passed Act 83 of 2019, establishing a state Nonprofit Security Grant Fund that has since distributed $25 million to synagogues, mosques, and other nonprofits. Muslim groups raised over $225,000 through a crowdfunding campaign to help pay for burials and medical bills. A total of $6.3 million was raised for survivors, victims' families, and responding officers. The building at Wilkins and Shady remains a place of memory and renewal, a congregation that chose its name well: Tree of Life.