A source close to the case said that the two men are being investigated for "spreading false news" and "offending" the head of government.
Judicial police arrested activist Bah Diakhate Monday after he recorded a video attacking Prime Minister Ousmane Sonko for an earlier statement on LGBTQ rights.
Preacher Cheikh Ahmed Tidiane Ndao was taken into custody on Tuesday, after also criticizing Sonko for what he said was complacency towards LGBTQ issues.
Sonko spoke about the issue during a visit last week by French far-left politician Jean-Luc Melenchon.
Religious figures and other activists in Senegal criticized Sonko for his perceived tolerance and for giving a platform to Melenchon to speak on the topic.
Homosexuality is stigmatized in the West African nation, where a law forbidding "acts against nature with an individual of the same sex" carries a penalty of up to five years' imprisonment.
Sonko warned last Thursday that insistence on LGBTQ rights in diplomatic relations and from multinational organizations could lead to "anti-Western sentiment."
Speaking to students in Dakar, Sonko urged Westerners to respect Senegalese culture and claimed there had "never been any persecution" of LGBTQ people in his country.
He suggested that same-sex relationships were "not accepted, but tolerated."