He was heard commenting about the 46-year-old judge’s beauty regime as he praised an act after they had performed.
Simon, 57, lauded schoolgirl magician Issy Simpson but seemed to give a back-handed compliment to Amanda in the process.
“I love the beginning of the trick when you become younger. You use magic, you use botox, the same effect,” he told Amanda with a little grin.
However, Amanda was clearly angry with him as she was left open-mouthed in shock at his words.
She almost threw her glass of water over Simon as he continued to talk about the Harry Potter magician’s incredible talent.
“I don’t know how you did that but you are a little star, aren’t you?” Simon told Issy, ignoring his fellow judge.
Luckily, Amanda managed to regain her composure after Simon gave her a look and simply said “no” as she put down the glass.
Amanda Holden lashed out at Simon Cowell on Britain's Got Talent
Amanda Holden tried to throw water over Simon Cowell on Britain's Got Talent
I love the beginning of the trick when you become younger. You use magic, you use botox, the same effect
This evening saw the second Britain's Got Talent live semi-final show take place as the eight-year-old gave another performance that felt positively magical and had a narrative that J.K. Rowling could have written.
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During the performance Issy asked the judges to pick a random celebrity, a favourite time of day, some change from their pocket and a number on a scrap of paper.
The trick was nearly ruined after judge Alesha Dixon read out the wrong number but David Walliams quickly corrected her and saved the day.
Amanda Holden was left shocked by the botox comment on Britain's Got Talent Britain's Got Talent: Most memorable moments Mon, May 1, 2017
Britain's Got Talent: The best and worst moments from the ITV talent show.
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Dance act Just Us get the golden buzzer
Alesha Dixon nearly ruined Issy Simpson's trick on Britain's Got Talent
Alesha, 38, accidentally read the number 30, 0517 as 30, 0515 which would have undermined the end of the trick because the digits corresponded to today’s date.
"I messed up because I was so mesmerised," she said defending herself but David, 45, made fun of her, saying that she might have had a pre-show tipple.
Yesterday Simon nearly ruined DNA's performance when he was having trouble locating a word in a dictionary.
Britain’s Got Talent continues on ITV tomorrow night at 7.30pm.