Mr Johnson claimed their stay was provided by David Ross, a donor to the Conservatives who co-founded the Carphone Warehouse mobile phone retail chain.
The spokesman later said: "Mr Ross facilitated accommodation for Mr Johnson on Mustique valued at £15,000".
Shadow Cabinet Office minister Jon Trickett has penned a letter to Kathryn Stone requesting she look into details of Boris Johnson's luxury holiday, which took place over the new year.
The Caribbean trip was also the first holiday Mr Johnson declared since going to Saudi Arabia in September 2018.
The Prime Minister visited St Vincent and the Grenadines with girlfriend Carrie Symonds between Boxing Day and January 5, 2020.
It emerged Mr Ross had used millions of pounds worth of Carphone Warehouse shares as collateral against personal loans without informing the company's other directors - a potential breach of City rules at the time.
In a statement to the Daily Mail, a spokesman for Ross dismissed Johnson's declaration, calling it "a mistake".
Labour, however, has stepped up its calls for a Westminster inquiry into how the PM came to enjoy the free provision of a five-figure villa.
British finance minister Sajid Javid resigned on Thursday, a surprise move that underlined Prime Minister Boris Johnson's desire to tighten his grip on government by jettisoning a minister who refused to toe the line.
"This was a benefit in kind from David Ross, a friend and former colleague".
A spokesman for the businessman said: "Boris Johnson did not stay in David Ross's house".
Mr Johnson faced criticism over his holiday for not returning sooner, after the U.S. killing of Iranian general Qasem Soleimani raised tensions in the Middle East.
Shadow foreign secretary Emily Thornberry accused the Prime Minister of "sunning himself" while leaving Cabinet Secretary Sir Mark Sedwill to chair three emergency Cobra meetings about the assassination.
He said: "Boris wanted some help to find somewhere in Mustique, David called the company who run all the villas and somebody had dropped out".
It provided Johnson with a break after the election campaign which saw him win a Conservative landslide.
"So Boris got the use of a villa that was worth £15,000, but David Ross did not pay any monies whatsoever for this".
In a declaration published on Wednesday, Mr Johnson announced in the MPs' register that he had accepted "accommodation for a private holiday for my partner and me, value £15,000" and named Mr Ross as the financial provider.
A No. 10 spokesperson said the Prime Minister had followed "all transparency requirements".