The Waratahs slumped to a 27-26 loss to their South African bogey side the Cheetahs and Barnes' latest setback only compounded their misery at Allianz Stadium.
Making his first appearance of the season after missing the opening month of the competition with a knee injury, Barnes appeared to get hurt while making a try-saving tackle on giant Cheetahs lock Lodewyk de Jager in the 17th minute.
With Barnes off contract at the end of the season and weighing up his future amid rumours he will take up a big-money deal in Japan, the 26-year-old might even have played his last game in Waratahs colours if the injury proves serious.
NSW's defeat was their third straight and the Tahs also take a dubious place in the Super Rugby history books as the first team to lose three times in a row to the Cheetahs.
Adding insult to injury, former Waratahs halfback Sarel Pretorius was the architect behind the South Africans' triumph, putting lively winger Raymond Rule over for the match-winning try 10 minutes from fulltime.
A 40th-minute try to centre Adam Ashley-Cooper had given NSW an unlikely 20-17 lead at the break and the Tahs continued to hold sway for most of the second half.
But after dominating possession and territory for the bulk of the match, the Cheetahs eventually broke through to back up their win last week on the road against the Highlanders.
Things looked ominous for the Waratahs from the moment Pretorius dummied and broke clear in the opening seconds of the match.
Centre Robert Ebersohn benefited from a lovely chip and regather from winger Willie Le Roux to score the opening try after just 54 seconds.
The Cheetahs were in again in the 17th minute when Rhule stepped his way over for the first of his two tries and five-eighth Johan Goosen again converted.
Two penalty goals from halfback Brendan McKibbin kept the Waratahs in touch at 14-6 before winger Peter Betham ran a nice line from a lineout win and took a short ball from McKibbin and beat two defenders to score under the posts.
McKibbin's conversion left NSW trailing by a point but, after Goosen added a penalty to nudge the Cheetahs four points ahead, the Waratahs snatched the halftime lead when Ashley-Cooper touched down on the stroke of halftime.
Goosen and McKibbin traded second-half penalty goals before Goosen's conversion of Rule's second five-pointer restored the Cheetahs' advantage.
Another McKibbin penalty in the 73rd minute reduced the deficit to one point but the Waratahs were unable to bridge it.
Earlier, the Hurricanes prevailed in a tight contest for a second week in a row, downing the Highlanders 23-19 in Dunedin.
One-point winners over the Crusaders last week, the Hurricanes outscored the Highlanders three tries to one.
But they needed two late Beauden Barrett penalties to settle a high-tempo contest in which the lead switched back and forth.
The result left the Highlanders winless after three matches and they also lost both their hookers during the match.
Skipper Andrew Hore went off within the first 10 minutes with a rib injury, and substitute Brayden Mitchell followed him just before fulltime.
The Hurricanes' third try of the night, from halfback AJ Perenara, nudged them ahead 17-16 early in the second half.
But Highlanders five-eighth Colin Slade landed his fourth penalty to get his side back in front, before Barrett slotted his two decisive goals, the second from 45 metres out and on an angle.
Earlier, in an opening spell in which both sides were able to recycle possession for long stretches, the Hurricanes were first on the board after keeping the ball for 13 phases.
The Highlanders, as they did at other times through the opening half, produced some tenacious defence, but their line finally broke as Smith went over.
The home side responded with two Slade penalties before the Hurricanes again dotted down, this time via prop Ben Franks off a strong Victor Vito run.
The Highlanders spent much of the opening spell in their own half and also came under pressure at scrum time, but they managed to hit the front before the interval.
From a scrum move, winger Hosea Gear, who was lively throughout, managed to get past some soft defence to touch down, with Slade adding the extras to put the Highlanders up 13-12.