Caesars Windsor laid off 38 unionized employees Monday.
The employees affected are from the food and beverage, culinary and resort operations departments.
Thirty full-time and eight part-time staff positions have been eliminated, offset by four part-time postings.
The casino said it needs to make operations more efficient.
"Business trends are optimistic, but we continue to have the obligation to find efficiencies in the business. Increased competition in Ohio, border issues, the smoking ban and exchange rate continue to be ongoing challenges our property faces," Caesars Windsor president and CEO Kevin Laforet said in a media release.
The majority of positions eliminated were servers, including some in Artist Cafe and Legends Sports Bar and Lounge.
"It's about operations and balancing our cost with our revenues," said Jhoan Baluyot, manager of public relations and communications at Caesars Windsor.
The employees were notified of the layoffs Monday morning. Depending on seniority, they have the ability to bump within the casino.
On Friday, Mayor Eddie Francis raised concerns about Caesars partnering with Rock Financial in purchasing Greektown Casino and potentially "cannibalizing" Caesars Windsor.
"We know a large portion of our clientele comes from the across the river. We want to ensure our Caesars property in Windsor continues to be viable," Francis told CBC News Friday. "It’s equally important that [the OLG] be aware of what’s happening across the river and stay on top of it for the benefit of the facility and from a community perspective."
Michael D'agnolo of the CAW, which represents hourly employees at Caesars Windsor, said jobs could have been saved if Ontario had single-event sports betting.
"It's concerning that we're looking at layoffs right now when we have something in front of the Senate called Bill C 290, single-game sports betting," D'agnolo said. "If this would have been passed right now, we'd be looking at hiring, not laying off.
"With these senators sitting on it for so long, I feel that they're putting these workers in this predicament."
On Friday, Francis said single-event sports betting would "be a shot in the arm for tourism."
"It would certainly grow the gaming industry," he said. "The federal government and senate has the ability to provide a competitive advantage to the tourism industry. The fact the Senate hasn't taken steps to enact that legislation is a surprise."
In question period Monday, provincial NDP finance critic Michael Prue called on the government to put a moratorium on the expansion of Ontario gaming in light of the financial impact facing Ontario communities.