Meanwhile, the visitors have failed to win any of their last three away matches in all competitions.
Livingston will be heading into their match with Ayr following the announcement that Gary Holt has resigned as manager after 27 months in charge.
Livingston have not won in five Scottish Premiership games but have had a brilliant Scottish League Cup campaign so far, having won all four of their Group H matches, scoring 15 goals – more than any other side in the competition.
The Lions faced Ayr in this competition last season, winning the match 2-1 at the Tony Macaroni Arena.
Another victory over Ayr would see the Lions reach the quarter-finals and win back-to-back matches against the visitors for the first time since 2000.
Ayr United's 6-5 victory on penalties against Stranraer was enough to take Mark Kerr's side into the last 16 of the Scottish League Cup.
The Honest Men also beat Annan Athletic and Albion Rovers as they went on to win three of their four Group H matches to finish top with eight points.
A 2-0 victory over Dundee is Ayr's most recent result in the Scottish Championship, which leaves them five points behind table toppers Dunfermline Athletic.
Prior to last season's League Cup defeat to Livingston, United have a good record against this weekend's opponents having won the previous five matches against them.
Victory on Saturday would see the Scottish Championship side reach the last eight of the competition for the second time in three years.
Livingston Scottish League Cup form: WWWW
Livingston form (all competitions): LLDWWL
Ayr United Scottish League Cup form: WLWW
Ayr United form (all competitions): WDLDWW
Livingston goalkeeper Robby McCrorie is ruled out as he has tested positive for coronavirus.
Ayr United boss Mark Kerr could revert back to a 4-2-3-1 formation, which has been used during their League Cup campaign.
Livingston possible starting lineup:
Stryjek; Devlin, Brown, Ambrose, Serrano; Bartley, Holt; Robinson, Pittman, Forrest; Emmanuel-Thomas
Ayr United possible starting lineup:
Sinisalo; Houston, Roscoe, Baird, Reading; McCowan, Chalmers, Murdoch, Smith; Moffat; Anderson
We say: Livingston 2-1 Ayr United
Both sides have performed well in the Scottish League Cup this season, scoring a combined 23 goals. Livingston, who remain unbeaten in the competition, will be eager to put in a performance for their new interim bosses and should have enough to narrowly defeat their Scottish Championship opponents.