Having restricted the Proteas to 163 for six in St Lucia, England looked to be on the way to a second Super 8s win in three days - and a place in the final four - but slipped to a seven-run defeat.
The top order completely mis-read the assignment, labouring to 60 for three off 10 overs, and without Brook, England would have been staring at an even more damaging loss than their closing score of 156 for six.
His daring 53 from 37 balls briefly positioned his side as favourites during a 78-run stand with Liam Livingstone (33), but it was not to be. With 14 needed off the final over, he was caught magnificently over the shoulder by Aiden Markram off Anrich Nortje's opening delivery - a match-winning piece of handiwork from the South African skipper.
England will kick themselves for a lack of timing and intent earlier in their pursuit, with a paltry return of three fours and one six in their first 11 overs at the crease.
Jos Buttler's men now move on to Barbados to face the United States, where victory is not guaranteed to send them through. Instead it could come down to net run-rate once again should the West Indies beat South Africa in the group finale.
Having hunted down 181 against the Windies at the same ground two days earlier England made a strangely subdued start, clamming up with 41 for one from their first six. In-form opener Phil Salt had crunched the third ball of the innings for six but but was gone before the end of the second over, Reeza Hendricks with a wonderful catch at cover.
Jonny Bairstow had better luck when he was put down by Heinrich Klaasen but he did not cash in, cutting the wily Keshav Maharaj to the man at point. The left-arm spinner was causing a headache has he teased the batters through the air and made a second major breakthrough when an unusually subdued Buttler picked out the boundary rider at midwicket.
Between them Bairstow and Buttler had used us 40 deliveries for 33 runs, raising the rate for those that followed.
By the halfway stage England had come to a near standstill, with their plight only worsening as Moeen Ali's mistimed a pull off Ottneil Baartman and failed to make the distance.
Brook and Livingstone were left with 103 to get from 58 balls and, although the boundaries finally started to flow, that was only enough to keep heads above water. Needing a big over they took 18 off Kagiso Rabada in the 15th, Livingstone kicking off the surge with a booming six into the leg side before Brook helped himself to four fours in five balls,
A cut and a ramp off Rabada were followed by a pair of hearty drives off Nortje. Suddenly it was South Africa under pressure and Baartman wobbled badly.
He served up five full tosses in six balls try and utterly failed to nail a yorker as he leaked 21.
With 25 needed from three overs England had become favourites just in time for Livingstone to turn another full toss, this time from Rabada, to the catcher at fine-leg. Brook still had the game in his hands with six balls left but Markram's amazing catch closed things down.
Quinton De Kock had earlier threatened to run away with it, his quickfire 65 eventually going down as a match-winning effort.
He ignored a modest head-to-head record against Moeen to lift his fifth ball over long-off, took Jofra Archer for back-to-back sixes with a flick off the hips and a crunching drive and pulled Sam Curran furiously into the stands.
While Reece Topley allowed just 18 from his three overs with the new ball, South Africa ran up 45 in the same period from the Pavilion End.
De Kock took just 22 balls over his fifty, the joint fastest in the tournament, and was handed a reprieve on 58 when a slog sweep off Rashid carried to Mark Wood at deep square. De Kock disputed a low catch and, while England were visibly aggrieved, the not out verdict looked fair on replays.
England barely celebrated when Hendricks bowed out with 19 off 25 but could not contain their joy when De Kock nicked Archer's well-disguised cutter and Buttler leapt to take a one-handed stunner.
Klaasen was promoted to number three to lift a stalling run-rate but also fell foul of Buttler's alertness as he flung a low, flat throw into the base of the stumps for a run out at the non-striker's end.
David Miller added late impetus with a knock of 43 but he and Marco Jansen fell to another pair of first-rate catches by Brook and Curran as Archer struck with successive deliveries at the death.