They were arrested after a rally in Bulawayo where Mrs Mugabe spoke last Saturday, the state-owned Herald newspaper reports.
They have now been released on bail and have not yet commented on the charges.
The booing came amid a bitter battle between rival factions of the governing Zanu-PF party to succeed President Robert Mugabe, aged 93.
Mrs Mugabe leads one faction, while former Vice-President Emmerson Mnangagwa leads the other. He was sacked two days after the heckling and has since fled to neighbouring South Africa.
The four accused are said to be supporters of Mr Mnangagwa.
Meanwhile, an American satirist arrested last week for allegedly referring to President Mugabe as "a goblin" has been freed on bail due to what the judge called "an absence of facts".
Grace Mugabe is now the favourite to take over from her husband
The three men and one woman have been freed on bail of $50 (£38) each and banned from attending a forthcoming presidential rally in the capital Harare.
Prosecutor Jerry Mutsindikwa told a magistrate court that "the quartet, with others, allegedly sang the song 'into oyenzayo siyayizonda'" - the lyrics of which say "we hate what you are doing" in Ndebele - while Mrs Mugabe addressed the rally, the Herald reports.
Mrs Mugabe had been pushing for the removal of Mr Mnangagwa, referring to him as a snake that "must be hit on the head."
He has been a close ally of the president for 40 years, dating back to the 1970s war of independence in which Mr Mugabe rose to prominence and his removal makes Mrs Mugabe the favourite to succeed her husband.
She is now expected to be appointed vice-president at a special Zanu-PF congress next month.
Who is Grace Mugabe?
- Began affair with Robert Mugabe, 41 years her senior, whilst working as a typist in state house
- Mr Mugabe later said his first wife Sally, who was terminally ill at the time, knew and approved of the relationship
- Married Mr Mugabe, her second husband, in 1996 in an extravagant ceremony. They have three children
- Nicknamed "Gucci Grace" by her critics who accuse her of lavish spending
- Along with her husband, is subject to EU and US sanctions, including travel bans
- Controversially received a PhD in September 2014 after just two months
- Appointed head of Zanu-PF women's wing in 2014
- Accused by a South African model of assault in August 2017