DFDS, which operates the roll-on roll-off (ro-ro) service from Rosyth in Fife to Zeebrugge in Belgium, admitted it would face a "big challenge" in retaining the route in 2015, when new European regulations limiting sulphur dioxide emissions from shipping come into force.
The Road Haulage Association and Freight Trade Association (FTA) both said its loss would mean HGVs carrying export goods from the north of Scotland to ports in England instead of Rosyth.
"The A1 to Newcastle would end up like the A9 is now, log-jammed with lorries travelling on long stretches of single-carriageway road without many opportunities for overtaking," said Phil Flanders, RHA director for Scotland and Northern Ireland.
Chris MacRae, the FTA's head of policy for Scotland, said increased emissions from lorries and worse congestion on UK roads would result if the Rosyth-Zeebrugge service closed.
This could lead to freight organisations sending goods the length of the UK in order to reach the continent, he added.
"You would see trucks heading south to Teesport and, daft as it sounds, they could end up going all the way down to the Channel ports because it would be cheaper to do it that way," he said. "You would see a huge amount of traffic on the A1."
A decline in use led DFDS to end passenger ferry services between Rosyth and Zeebrugge in 2010, retaining a freight-only service three times a week.
A confidential report earlier this month, commissioned by Edinburgh-based transport partnership SEStran, found DFDS had been losing money on the route since it converted to freight-only and the volume of trucks and other vehicles it carried in 2011 fell by 5% to 43,000. The traffic this year was expected to be "possibly marginally lower", the report found.
Professor Alan McKinnon, an expert on freight at Heriot-Watt University, recently warned the Rosyth-Zeebrugge ferry had not attracted enough traffic to be commercially viable and was unlikely to survive the introduction of tougher sulphur dioxide emissions regulations, which will push up fuel costs. "The Rosyth-Zeebrugge service may not survive," he said.
A spokesman for DFDS said there were currently "no plans for winding up" the Rosyth-Zeebrugge route. However, he added: "It cannot be denied the sulphur-emission regulations that come into force in 2015 constitute a very big challenge for many of our routes, and not only DFDS's routes, but all ro-ro and passenger routes in the Sulphur Emission Control Area – in particular routes that run parallel to roads."