Coming just over two months before national elections, the raid adds to the woes of the ruling African National Congress (ANC), which is struggling in the polls amid accusations of graft and mismanagement.
The National Prosecuting Authority (NPA) said members of a top investigative team carried out a "search and seizure operation" at Nosiviwe Mapisa-Nqakula's residence, a high-end property in an eastern suburb of Johannesburg.
Parliament said the operation was linked to a probe into alleged corruption during Mapisa-Nqakula's tenure as defence minister.
"The Speaker steadfastly upholds her strong conviction of innocence, and reaffirms that she has nothing to hide," it said.
"She has welcomed investigators into her home, cooperating fully during the extensive search that lasted over five hours."
The raid follows local media reports accusing the ANC veteran of soliciting 2.3 million rand ($121,000) in bribes from a former military contractor.
Mapisa-Nqakula served as defence minister between 2014 and 2021 before being appointed speaker in a move that drew much criticism from the opposition.
At the time, she had recently come under fire for perceived incompetence in responding to a spate of deadly unrest that killed more than 300 people.
South Africa's leading opposition party, the Democratic Alliance (DA), on Tuesday called for her to step down.
"Her continued presence in office undermines the integrity of the legislative institution," said DA chief whip Siviwe Gwarube.
Mapisa-Nqakula, who as speaker is fifth in the line of presidential succession, is the latest in a string of senior ANC politicians, including the president and vice president, to be embroiled in graft scandals.
South Africans head to the polls in national and provincial elections on May 29.
In power since the advent of democracy in 1994, the ANC is expected to see its share of the vote drop below 50 percent for the first time.
This might force it to form a coalition to remain in power.
Mapisa-Nqakula is "profoundly disturbed" by the allegations against her, parliament said.
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