The accident occurred just before 7 p.m. near mile marker 74 on I-81, according to the Tennessee Highway Patrol.
The driver of the tractor-trailer — Carl Russell Osburn, 54, of Princeton, W. Va. — died as a result of the crash.
Also killed in the accident was 81-year-old Dennis Darrell Dawson, of Begley, W. Va. Dawson was driving a 2012 Toyota Camry.
Dennis Dawson's wife and passenger — Connie Sue Dawson, 75 — was seriously injured in the crash.
All three individuals were reportedly wearing safety belts when the accident took place.
A preliminary THP incident report shows the two vehicles were traveling north in the right lane of I-81 when Osburn's truck — which was loaded with tires — struck the back of the Dawsons' vehicle.
The collision reportedly caused both vehicles to cross two lanes of traffic and strike a concrete bridge pillar.
As a result of the impact, Osburn was ejected from his vehicle and into the southbound lane of I-81.
At that point, authorities said Osburn was run over by a southbound vehicle.
However, THP Sgt. Bill Miller said that the investigation process has determined the driver was killed as a result of the injuries sustained during the original crash.
The motorist who struck Osburn was not injured, but was taken into custody by THP for driving on a suspended license.
Both the tractor-trailer and the Dawsons' Camry came to rest on the left shoulder of the interstate, the report stated. The sedan was reportedly wedged between the concrete pillar and the trailer's left rear tandems.
The trooper working the crash indicated in his report that seat belts would not have made a difference in the two fatalities.
The investigation of the wreck is ongoing.
The Bristol Police Department and Bristol Fire and Rescue were joined by state troopers in responding to the crash, which shut down both lanes of I-81 for several hours Saturday night. The THP's Critical Incident Response Team was also dispatched to the scene to investigate.