Ms Machado, a former congresswoman, led a major street protest against President Maduro's government in January.
She dismissed the accusations as a charade designed to silence her.
The alleged plot came in a series of emails which Ms Machado says are fake.
Ms Machado said the charges were designed to distract Venezuelans from a growing economic crisis.
Officials produced the emails in the midst of months of street protests.
They said they contained conversations between Maria Machado and US State Department officials discussing a plot to overthrow the Venezuelan government.
Ms Machado said the messages used her old email accounts and had been manipulated and were fabricated.
She said on her social media account that the conspiracy charges were in retribution for demanding a new leadership at the state elections council.
Maria Machado had helped lead demonstrations which had initially been started in January in the western state of Tachira by university students.
They were protesting against the high rate of crime on campuses and the country's struggling economy.
She was expelled from the National Assembly in March after accepting an invitation from Panama to speak before the Organization of American States (OAS), where she gave her account of the wave of unrest which spread through Venezuela earlier this year.
The President of the National Assembly, Diosdado Cabello, has accused her of inciting violent protests in which more than 40 people died.
Before her court appearance, she said: "Our protest movement has always been peaceful in its essence. Violence is what the regime does to frighten people and de-motivate citizen protest."
She said she would continue to support all types of protests in the country against what she described as "daily abuses committed by the government".
Since narrowly winning an election last year to succeed his mentor, the late President Hugo Chavez, President Nicolas Maduro has said there have been five assassination attempts against him and more than a dozen acts of sabotage and conspiracy.
Conspiracy carries a prison sentence of between eight and 16 years in Venezuela.
Correction 27 March 2015: This report has been amended to clarify the reason for Ms Machado's expulsion from the National Assembly.
Venezuela's chief prosecutor has launched an investigation into leading opposition figure Maria Corina Machado over an alleged plot to assassinate the President, Nicolas Maduro.