Both Charles (hamstring) and his midfield partner Ali McCann (arm) will miss the final two games of the campaign through injury, as will defender Brodie Spencer, while Ethan Galbraith is suspended for the game in Kosice.
The upshot is O’Neill will be forced to field a much-changed team from the one that defeated Slovakia 2-0 in Belfast last month, with Isaac Price the only one of the usual midfield four available.
Shea’s younger brother Pierce Charles – Sheffield Wednesday’s highly-regarded goalkeeper – will also miss out due to injury.
O’Neill said: “Shea’s a huge one because since I came back in he has only missed one game and that was through suspension. It was the Finland game after being at the red card here against Slovenia.
“In that period of time, I’ve seen him grow from a young development player at Manchester City to being a key focal point of our team and obviously a key focal point of his club side, Southampton, as well. It’s a huge blow.
“We’ve had periods without Ali in the past. Ali’s been unlucky with injury and this is another example of that. The injury was so innocuous. For someone to pull your arm and it ends up getting broken. It’s incredible, really, and another big blow.
“We missed Brodie in September with a muscle injury and now he’s done his metatarsals. All three lads will be a big loss to us on top of what we already had with Pierce Charles out as well.
Michael O’Neill discusses injury situation ahead of crucial World Cup qualifiers
“There’s others around the periphery like Jordan Thompson, Dale Taylor and Jamal Lewis who have missed out during this campaign.
“The squad has been tested I think and they’ve stood up to that and showed great character. They’ll have to show that again.”
Earlier this month, then-Southampton manager Will Still confessed that Shea Charles started a League match will struggling with a hamstring injury, only to be subbed off later in the game.
O’Neill said: “Sometimes players play for their clubs with niggles and we’re not in control of that situation. It is frustrating because if a player gets injured they’re going to miss two games for us. But it is out of our control.”
O’Neill is now faced with a conundrum with both deep-lying midfielders – Charles and McCann – out of action. It’s likely that veteran George Saville will start in Kosice, but the spot beside the Luton Town man in the middle of the park is very much up for grabs.
Kilmarnock’s Brad Lyons is an option, as is Mansfield Town youngster Jamie McDonnell.
However, O’Neill may move Crystal Palace man Justin Devenny into the middle from left-wing back and rearrange his defence.
O’Neill explained: “We’ve talked about it already amongst the coaching staff. Just in terms of our preparation, we feel we have the right players to slip in there.
Northern Ireland boss Michael O’Neill
“We would probably have to be a little bit creative with our selection in the midfield area or we would maybe quite inexperienced in that area.
“There’s a balance between experience and asking players to do something a little bit different in this game. If you look at Justin Devenny, for example, he is essentially a midfield player. We’ve asked him to play as a left wing-back and he’s done that exceptionally well.
“Paddy McNair has played in midfield for us over the years in the past as well, so that’s another option.
“There are different things that we can look at.”
One new face in the Northern Ireland squad is former Coleraine midfielder Patrick Kelly, who has impressed at Barnsley this term after joining from West Ham United during the summer.
O’Neill said: “He made a brave move. The chance he could have gone on loan in the summer, but he felt he’d be better going on a permanent basis.
“We’ve watched him consistently, felt he played well in the 21s, so he has earned his place his place in the squad, as has Brad Lyons who is playing well for Kilmarnock.”
Northern Ireland play Slovakia in Kosice on Friday November 14 before hosting Luxembourg on Monday November 17. A second-placed finish will guarantee Michael O’Neill’s men a place in March’s World Cup Play-Offs.