Isaias is the first hurricane of the Atlantic season and was expected to usher in dangerous conditions in the Florida Panhandle, southern Alabama and southern Mississippi when it crosses the coast late on Friday.
Heavy rain and flooding were possible into the weekend as far north as Tennessee and North Carolina.
The category two hurricane is most likely to strike along the Alabama-Florida state line and bring life-threatening storm surge across Pensacola bay, according to the National Weather Service.
But there is a small chance it could hit further west and have a major impact on Mobile, Alabama, or further east and have a strong effect on the Florida cities of Pensacola and Destin.
Meanwhile, Hurricane Simon, which formed as a tropical storm on Wednesday off Mexico and became a hurricane late on Thursday, was forecast to strengthen into a category four hurricane.
Isaias had maximum sustained winds of 165km/h and heading north-northeast, according to the hurricane centre, and centred about 370km south of the mouth of the Mississippi River.
A hurricane warning was in effect from Ocean Springs, Mississippi, to the Bay-Gulf County line in Florida, the hurricane centre said.
A storm surge warning was in effect from the mouth of the Mississippi to the Suwannee River.
Coastal communities moved quickly to prepare on Thursday, as Alabama Governor Kay Ivey ordered non-residents to evacuate.
The governors of Florida and Alabama declared states of emergency for dozens of counties, with Florida's Ron DeSantis saying about 5000 National Guard troops were expected to mobilise by Friday.
Water from a storm surge and high tide could reach heights of 1.5 to two metres in parts of Alabama and Florida.
Isaias also could dump up to 38 centimetres of rain on coastal areas and spawn tornadoes overnight on Friday into Saturday from Alabama to the Florida Panhandle and southwestern Georgia.
The hurricane is expected to quickly weaken after coming ashore.
Isaias developed more than four months into the Atlantic hurricane season.
The Atlantic hurricane season has been unusually quiet because of a strong El Nino, which disrupts storm formation over the Atlantic.
Storm activity has been above average in the Pacific, where Hurricane Simon formed.
Farther north, Tropical Storm Rachel was expected to reach Baja California, Mexico, and southern California at the weekend.
