Mr McCluskey, who is the general secretary of Unite, called Tom Watson a “f****** disgrace” after the MP criticised the party’s general secretary over the handling of antisemitism complaints.
Mr Watson has faced backlash because Jennie Formby is undergoing chemotherapy for breast cancer.
Speaking at the Durham Miners’ Gala, Mr McCluskey told thousands: “I have a simple message for Tom Watson and his pals in the media – a simple message to Tom and his pals: You should f****** well be ashamed of yourselves.
“Jennie, our message to you is that the Durham Miners’ Gala stands with you.”
The comment drew a huge cheer from those gathered at the Gala.
After the speech, he tweeted: “I’ve said it today at the Durham Miners’ Gala so I’ll repeat it here. Attacking a woman going through chemotherapy – @tom_watson you are a f****** disgrace. @JennieGenSec.”
Mr Watson had written to Ms Formby calling for the party to publish its full submission to a watchdog investigating antisemitism claims, as well as raising allegations she had deleted emails relating to cases – a suggestion Ms Formby denies.
In her reply, she said she was “very disappointed” about his approach and accused him of abusing his position, while a number of MPs leapt to her defence.
Emma Dent Coad, Kensington MP, even retweeted people calling for Mr Watson to step down.
Mr Watson’s letter came as the fallout from a BBC Panorama documentary into Labour’s handling of antisemitism continued.
Eight people told the programme that Labour’s director of communications, Seumas Milne, and Ms Formby interfered with investigations.
Four of them broke non-disclosure agreements to speak out, including former party general secretary Iain McNicol.
But a party spokesman slammed the programme, saying it “engaged in deliberate and malicious representations designed to mislead the public”.
Party leader Jeremy Corbyn spoke at length about the “many, many inaccuracies” in the Panorama programme during his speech at the gala.
He said: “The programme adopted a pre-determined position on its own website before it was broadcast.
“We’ve made very clear what our processes are.
“Our party members do have the right to be heard if they’re accused of anything and our party staff have a right to be supported and they are supported.”
Mr Corbyn said he would co-operate with the Equalities and Human Rights Commission (EHRC) inquiry into allegations, and said: “Antisemitism is a poison, it is vile, it is wrong.”
Sky News has contacted Mr Watson for a comment.