Quins will be kicking themselves back down the motorway after they had Gloucester in trouble a few times in the second half but could not get the tries to recover from an early setback.
Wing Charlie Sharples, full-back Jonny May and flanker Akapusi Qera grabbed Gloucester's tries, with fly-half Freddie Burns booting four penalties and a conversion.
As well as Chisholm's tries, Quins' other points came from fly-half Nick Evans' boot with three penalties and two conversions.
After Saracens' surprise loss to Worcester on Friday night, Harlequins were left kicking themselves with the knowledge before kick-off that victory at Kingsholm would open up a nine-point gap on their London rivals.
But they started the match in the worst possible way to go 10 points down after as many minutes.
It was former England winger James Simpson-Daniel who became a real thorn in the Quins' side during those early stages.
Just moments after Burns had booted a monster 49-metre penalty to give the home team the lead, Simpson-Daniel almost got a try when he dashed downfield only for an excellent tackle to bring him down just short.
However, set free on the right, the flyer superbly dummied the Quins defence to put Sharples in for a converted try.
Evans added to the 159 points he has scored this season with two penalties that brought Quins back to within four points.
It was turning into a lively, free-flowing game, with both teams trying to play open rugby.
But Quins' midfield defence, mainly responsible for the opening try, let them down again when May, who has been highlighted as a hot England prospect by national boss Stuart Lancaster, squeezed through some weak tackling and dashed 45 metres to score unopposed on the left.
Ex-England wing Ugo Monye was lucky not to find himself in the sin-bin as half-time approached when he high-tackled Sharples.
Burns added a second penalty to make it 18-6 but Quins could have gone into the break just five points down when Evans put in a delicate grubber kick 10 metres from the line that Chisholm grasped for a converted score. However, they committed an error and Burns booted his third penalty of the half with the last play.
The 21-13 half-time deficit may have worried Quins bosses, who have seen their side stutter in the Premiership since Christmas after recording 10 straight league wins that took them to the table summit.
Burns and Evans swapped penalties and the hosts they went well into the lead when big flanker Qera finished off a move across the backs in the 58th minutes.
But Quins' pack, led well at number eight by skipper Nick Easter, tried their hardest to grab a second try they deserved.
Their golden opportunity turned to dust, though, when, piling the pressure on the Gloucester line and just two metres away from that elusive try, the Cherry and Whites turned the ball over, hoofed downfield and threatened to get their fourth bonus-point score.
With the game gone, Quins didn't have a sniff of victory again and the only disappointment for the home side was that they did not get the try bonus point they were close to on a couple of occasions and that they let Chisholm in for his second score, a length-of-the-field effort from his own 22, in the last movement of the match.
That allowed Evans to convert from the touchline and gain the losing bonus point, which was the only crumb of comfort for the frustrated Londoners.
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