Today, Bank of America, American Airlines, Uber and other companies joined the list of signatories, bringing the number to 86.
These companies, along with other opponents of the law, have asked North Carolina Gov. Pat McCrory and state lawmakers to repeal the law, formally known as the Public Facilities Privacy and Security Act. Last Wednesday, the North Carolina General Assembly passed the bill and McCrory signed it into law that day.
The law bans people from using bathrooms that don't match the sex indicated on their birth certificate.
Proponents, like the North Carolina Family Policy Council, explain that "state and local governments must abide by a statewide policy regarding the use of single-sex multiple occupancy bathrooms by individuals based on the biological sex stated on their birth certificate (which, by law, can be changed if an individual undergoes sex reassignment surgery)."
The council argues on its website that the law "does not impact the ability of businesses to adopt their own internal employment, non-discrimination, or bathroom policies."
ESPN, which has considered the Charlotte Motor Speedway for the site of future X Games events, said it's evaluating its options. The Walt Disney Company is the parent company of ESPN and ABC News.
On Tuesday, city leaders in Atlanta asked the NBA to consider moving the 2017 All-Star Game to their city from Charlotte because of the new law.
Lawmakers in Georgia are also facing backlash after voting in favor of a bill that opponents say limits the rights of LGBT individuals. Last week, 34 entertainment industry players wrote to Georgia's Gov. Nathan Deal about the state's Free Exercise Protection Act, saying they "will plan to take our business elsewhere if any legislation sanctioning discrimination is signed into law." Georgia's bill would allow clergy to refuse to perform marriage rites that violate their religious beliefs. It would also allow churches and religious groups to decline services to someone if doing so it conflicts with their faith. The governor has indicated that he will veto the bill.
ABC News' Avianne Tan contributed to this report.