Hughes announced his decision to call time on his international career last month but was hoping to lead his country out for a final time at Windsor Park on Friday before a high-profile final appearance against the Azzurri four days later.
End of the road: Hughes' international career is over
But the 31-year-old was forced off after 20 minutes of Fulham's Barclays Premier League match against Blackburn on September 11 and has not played since. The former Newcastle and Aston Villa centre-half finishes with 79 caps, having made his debut in 1998, scoring his only goal in the 4-0 defeat of the Faroe Islands in August.
Hughes will now hope to extend his top-flight career for as long as possible under Cottagers boss Martin Jol, while Northern Ireland face up to their latest batch of injuries.
Keen to stay: The Fulham defender is holding out for a new contract at Craven Cottage
Manchester United defender Jonny Evans, who has been a regular at left-back under current manager Nigel Worthington, is the obvious replacement for Hughes in the middle but he could also miss the next two matches with an ankle injury.
He played the full 90 minutes for United in their win over Norwich at the weekend, but is rated as doubtful for the final qualifiers of what has turned into a disappointing campaign for Worthington's men.
Blackpool's Craig Cathcart is the next likeliest candidate to partner Gareth McAuley in the centre, creating a possible vacancy at right-back and uncapped Preston full-back Conor McLaughlin has been added to the squad as a result.
Under-pressure boss Worthington, whose contract is up at the end of the current campaign, is also struggling for options in attack, with Millwall's Josh McQuoid (virus) and Derby's Jamie Ward (hamstring) also pulling out of the original 23-man party.
Highly-rated Rams forward Ward made his debut as a late substitute in the win over the Faroes but has now withdrawn from the last two squads through injury.
Worthington must also choose someone to take the armband in Hughes' absence, with the likes of McAuley and Chris Baird among the candidates.
Rangers skipper Steven Davis is likely to be the favourite, though, as he is a good long-term bet for the job and could be handed the role for the Estonia clash in recognition of his 50th cap.
Retiring Hughes to lead Northern Ireland into final two international matchesHughes to call time on his Northern Ireland career at the end of Euro 2012 campaign