Unifor says the new strike deadline is 5 p.m. Monday.
Notice of the new deadline was served to the Seaway on Friday morning during ongoing contract negotiations in Cornwall, Ont.
Unifor National Representative Joel Fournier says the union remains hopeful that a deal can be reached.
The union says talks are expected to continue through the weekend under a media blackout, and that the two sides have been negotiating since Tuesday.
Five Unifor locals along the seaway from Niagara to Montreal announced Tuesday they were serving 72 hours' strike notice along the waterway.
Under federal labour law, 72 hours' notice must be given of any potential strike or lockout.
Labour Minister Kellie Leitch said earlier this week that the federal government was "disappointed" to hear that the St. Lawrence Seaway Management Corp. and Unifor have not come to terms on collective agreements.
"I strongly encourage both parties to continue negotiating to find a solution that will benefit everyone," Leitch said in a statement Wednesday.
"The best solution in any dispute is always the one that the parties reach themselves."
The union says one of the key issues in contract talks is staffing levels at the locks as the seaway moves to hands-free mooring, eliminating staff currently working on the locks.
The union is calling for minimum staffing levels on the locks to deal with emergencies.
The strike notice was served at the resumption of contract talks in Cornwall on Tuesday, the first time the two sides had met in months.
A federal mediator is assisting with negotiations after Unifor filed for federal conciliation in August. The workers earlier voted 96 per cent in support of a strike.
The five union branches along the St. Lawrence Seaway — Locals 4212 and 4211 in Niagara and Cornwall, Locals 4319 and 4320 in Montreal and Local 4323 in Iroquois, Ont., — have about 460 members.
The St. Lawrence Seaway, which extends from Montreal to mid-Lake Erie, includes 13 Canadian and two U.S. locks.