Donald Trump did not appear in person at the Reed Arena in Texas, but recorded a video message which was played to concert goers.
The existing President spent much of the weekend at his golf course in Virginia.
He said his wife Melania "and I want to express our deep gratitude for your tremendous assistance.
"This wonderful effort reminds us that we truly are one nation under God, all unified by our values and our devotion to one another."
Lady Gaga also made a surprise appearance, and tweeted: "Nothing more beautiful than everyone putting their differences aside to help humanity in the face of catastrophe. #OneAmericaAppeal"
The fundraising appeal has so far raised $31m (£24m) for the relief effort.
This Atlantic hurricane season has been one of the worst in recent years. Irma killed 87 people in the US and its territories, while the US death toll from Maria now stands at 24, all in Puerto Rico.
Hurricanes Harvey and Irma are expected to cost the US between $150 (£113) and $200bn (£151bn) in combined property damage, according to the Moody's credit ratings agency.
In Puerto Rico, hundreds of thousands of people are still without running water and electricity.
President Trump has been criticised for his response to the hurricane in Puerto Rico, including from the Mayor of San Juan, Carmen Yulin Cruz.
Ms Cruz recently issued a public plea to Donald Trump to do more to help the country.
“I ask every American that has love, and not hate in their hearts, to stand with Puerto Rico and let this President know we WILL NOT BE LEFT TO DIE,” she wrote on Twitter.
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