Deputy Tourism Minister Frederico Silva da Costa and 37 other officials from the ministry have been arrested on corruption charges as police allege they conspired to divert public money for private gain.
The officials were arrested in the cities of Brasilia, Sao Paulo and Macapa.
Prosecutors said the suspects had awarded contracts at inflated prices to complicit firms that often were not qualified to carry out the work.
Costa is the third high-ranking official to leave his post over corruption allegations since Rousseff took power in January.
The arrests come in the wake of allegations of corruption at the ministries of agriculture and transport. Agriculture Minister Wagner Rossi has been asked to appear before an ethics commission to answer the allegations.
Last month, Transport Minister Alfredo Nascimento and more than 20 officials resigned over allegations of kickbacks at the ministry of transport.
And in June, the president’s chief of staff Antonio Palocci resigned after press reports questioned his rapid accumulation of wealth.