The allegations were made in the investigative journalism TV program Le Iene (The Hyenas), in an episode which aired on Sunday evening.
Former employee Salvatore Pizzo told reporters he worked without a contract at Ardima Srl between 2009 and 2010.
Pizzo also said that after he was hurt in a work accident, owner Antonio Di Maio told the construction worker to lie about how he had obtained his injuries so the incident would not be reported to the authorities.
Salvatore Pizzo on Le Iene
Luigi Di Maio was made 50 percent owner of the company along with sister Rosalba approximately two years after Pizzo worked for the outfit, according to the Le Iene report.
In a statement, Di Maio said he was happy that Pizzo had had the courage to come forward, but distanced himself from the allegations, saying he had no management role in the company at the time and no knowledge of the discrepancies detailed in the report.
He added that he and his father have in the past had a strained relationship.
“My father made mistakes in his life, and I distance myself from this behaviour, but he is still my father,” the deputy premier wrote in a Facebook post.
“Tomorrow I will hand over to the relevant documents to [Le Iene reporter] Filippo Roma, which I have requested from my father, and we will complete all the necessary checks on what Salvatore said in the programme.”
Relations between M5S and the press, which have deteriorated since the party came to power, reached new lows this month when its leadership launched a barrage of insults at journalists, with Di Maio characterising reporters as 'jackals' and M5S leader Alessandro Di Battista dismissing them as 'whores'.