In its lawsuit, Disney accused DeSantis and his supporters of illegally using the state government to punish a company for voicing an opinion that should be protected by free-speech rights.
Florida lawmakers passed legislation that ended Disney's virtual autonomy in central Florida where the Disney World theme parks attract millions of visitors each year.
In the action filed in federal court in Tallahassee, Disney said it aimed to protect Disney World's employees, guests and developers from "retaliation for expressing a political viewpoint unpopular with certain State officials."
"Disney now is forced to defend itself against a State weaponizing its power to inflict political punishment," the company said.
Last year, Disney's then-chief executive, Bob Chapek, said the company opposed a bill formally known as the Parental Rights in Education Act. Critics called it the "Don't say gay" law.
Disney's lawsuit alleges that a newly formed DeSantis-appointed tourist board violated the company's contract rights, and did so without just compensation or due process. The company is asking the court to declare Florida's legislative action unlawful.
DeSantis has argued that Disney, which employs roughly 75,000 people in Florida, had been enjoying unfair advantages for decades.
"We are unaware of any legal right that a company has to operate its own government or maintain special privileges," DeSantis spokesman Jeremy Redfern said Wednesday on Twitter.
The governor is currently traveling in Asia on a four-country trade mission.
Disney shares fell 1.4% to close at $96.61 on the New York Stock Exchange on Wednesday.
POLITICAL RISK
DeSantis’ clash with Disney has been a centerpiece of his speeches as he toured the United States ahead of his expected presidential bid. But as the battle has intensified, it has brought mounting political risk.
Former President Donald Trump, the favorite for the Republican nomination, has slammed DeSantis’ stance, saying on social media that the governor "is being destroyed by Disney" and warning that the company would reduce its investments in Florida.
Carlos Curbelo, a former U.S. Republican congressman from Miami, said DeSantis' attacks on Disney "made sense for a time."
"Now it’s coming across as petty and personal," Curbelo said. "Disney clearly detects that the governor is in a weaker position today and is going on offense for the first time in this conflict."
Before DeSantis appointees took over a state board that oversees Disney World, the company pushed through changes to the special tax district agreement that limit the board's action for decades.
Florida's new oversight body on Wednesday said Disney's plans for potential expansion of Disney World did not comply with state law, and declared that agreement void.
The Central Florida Tourism Oversight Board unanimously supported an attorney's findings of legal flaws in the developers' agreement Disney reached in February with a previous board, including a lack of proper public notice.
"What they created is an absolute legal mess," said board Chairman Martin Garcia. "It will not work."
Disney announced its lawsuit minutes later.
The tussle could boost DeSantis' support among U.S. Republican voters, a Reuters/Ipsos poll found, but also hurt him among the wider electorate.
Seventy-three percent of respondents - including 82% of Democrats and 63% of Republicans - said they were less likely to support a political candidate who backs laws designed to punish a company for its political or cultural stances.
The judge that will oversee Disney's case against DeSantis, U.S. District Judge Mark Walker, has struck down several laws that defined the governor's conservative political agenda, including statutes that sought to limit the speech of college professors, curtailed protests and restricted voting access.
