Sandra Lonardo, the minister's wife who heads the council of the southern Campania region for UDEUR, is accused of accepting favours from a hospital in Caserta, near Naples.
In a statement, Lonardo said: "I believe this is the bitter price that my husband and I are paying for our defence of Catholic values in politics, for the principles of moderation and tolerance against all fanaticism and extremisms."
A court document showed that Mastella is also under investigation.
The minister, who has insisted for months that he has been the target of a witch hunt, told parliament: "Now I realise that I am seen by some extremist fringes as an adversary to fight or even an enemy to beat down."
He said that he had "worked day and night to demonstrate my credibility and my good faith as a trustworthy interlocutor in the world of justice".
Mastella is the first minister to resign in the Prodi government and had already threatened to pull his party out of the fractious nine-party coalition over electoral reforms.
The reforms were given a boost on Thursday when Italy's constitutional court gave the go-ahead for a referendum on a new voting system.
If passed, the reforms would require parties to win 4 per cent of the national vote to secure parliamentary representation in the lower house and 8 per cent for the upper house.
Mastella's party had opposed the change, having won just 1.4 per cent of the vote in April 2006 legislative elections.