The US Treasury said the measures are designed to steer American travellers away from Cuban firms tied to the military and towards the communist island's fledgling private sector.
Much of the half-century-old US economic embargo against Cuba remains entrenched in law, but under former president Barack Obama federal authorities began to loosen some rules.
Ties had begun to warm, and in 2015 the countries' exchanged ambassadors for the first time since 1961.
But in June Trump appeared in Miami before a cheering crowd of Cuban-Americans, including veterans of the failed CIA-backed Bay of Pigs invasion, to vow to reverse Obama's measures.
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"We have strengthened our Cuba policies to channel economic activity away from the Cuban military and to encourage the government to move toward greater political and economic freedom for the Cuban people," he said.
Cuba criticised the move as marking a "resurgence" of the economic embargo.
"The measures confirm the serious setback that has taken place in bilateral relations as a result of the decisions taken by the government of President Donald Trump," Josefina Vidal, the head of North American affairs at Cuba's foreign ministry, told journalists.
Also on the list are five major holding companies with ties to Raul Castro's government or military, and which between them control much of the organised tourism sector.
The list goes on to list dozens of major hotels in Havana and several resorts, along with five Caribbean marinas, ten stores in touristy Old Havana and industries serving the military.
Two rum distilleries are on the list, along with a shopping mall, a photo developer and a real estate broker.
In addition to staying in privately-run accommodation and thus supporting small-scale capitalism, the travelers will be expected to engage in authorised exchanges with Cuban people.
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