Flores is married to President Nicolás Maduro, and replaced Maduro as President of the National Assembly in August 2006, when he resigned to become Minister of Foreign Affairs, with Flores becoming the first woman to serve as President of the National Assembly. The two had been in a romantic relationship since the 1990s when Flores was Hugo Chávez's lawyer following the 1992 Venezuelan coup d'état attempts and were married in July 2013 months after Maduro became president.
Her husband Maduro has one son, Nicolás Maduro Guerra, whom he appointed to senior government posts: Chief of the presidency's Special Inspectors Body, head of the National Film School, and a seat in the National Assembly, while Flores has an adopted son, Efraín Campos, who is her nephew from her deceased sister.
As the lead attorney for Chávez's defense team, she was instrumental in securing Chávez's release from prison in 1994 after his unsuccessful coup in 1992.
While serving as chair of the Political Command of the Bolivarian Revolution, Flores was part of the Tactical Command for the Revolution, an organization that ran the majority of Hugo Chávez's political machine. On 7 April, days before the 2002 Venezuelan coup d'état attempt, Flores along with Guillermo García Ponce and Freddy Bernal shared plans of using the Bolivarian Circles as a paramilitary force to end opposition marches and defend Chávez in Miraflores Palace by organizing them into brigades.
On 11 April while opposition marchers headed towards Miraflores Palace in protest, Bolivarian Circles gathered around the palace armed with rocks, clubs and molotov cocktails all within view of the National Guard that was stationed nearby. The Bolivarian Circles then participated in demonstrations that became violent.
A member of the United Socialist Party of Venezuela (PSUV), Flores replaced her husband Maduro as Speaker of the Assembly in August 2006, when he was appointed Minister of Foreign Affairs; she was the first woman to serve as president of the National Assembly (2006–2011). On 10 January 2007, Flores swore Chávez into office following the 2006 presidential election.
Flores is running for a seat in the National Assembly in Venezuela's 2015 parliamentary elections as a candidate for the Great Patriotic Pole. She said she would use her seat to defend the social rights of citizens and the achievements of the Bolivarian Revolution.
Upon Maduro's tight victory in the 2013 presidential election over Henrique Capriles, Cilia Flores became Venezuela's First Lady, a position that had long been vacant.
Flores was accused of nepotism with individuals claiming that several of her close relatives became employees of the National Assembly while she was a deputy. According to Tal Cual, 16 relatives of Flores were in an office while she was in the National Assembly. Flores responded to the reporters who shared the nepotism allegations stating it was part of a smear campaign, calling them "mercenaries of the pen". Both opposition and members of the government denounced the alleged nepotism calling it an injustice, with one PSUV member taking the allegations to Venezuela's Ministry of Labour. In 2012, relatives of Flores were removed from office though some received other occupations in the government a year later.
Flores' son, Walter Jacob Gavidia Flores, whose last salary through 2015 was less than $1,000, made multiple international trips in 2015 and 2016 on private flights costing approximately $20,000 per trip. Gavidia Flores spent most of his time in the United States, though he also took chartered flights to France, Germany, Malta and Spain.
On 10 November 2015, two nephews of Cilia Flores, Efraín Antonio Campos Flores and Francisco Flores de Freitas, were arrested in Port-au-Prince, Haiti by local police while attempting to make a deal to transport 800 kilograms of cocaine destined for New York City and were turned over to the Drug Enforcement Administration where they were flown directly to the United States. Campos stated on the DEA plane that he was the step son of President Maduro and that he grew up in the Maduro household while being raised by Flores. The men traveled to Haiti with Venezuelan diplomatic passports but did not have diplomatic immunity according to former head of DEA international operations Michael Vigil. The two were previously monitored and filmed by the DEA between October and November 2015 after they contacted a DEA informant for advice on trafficking cocaine and brought a kilogram of cocaine to the informant to show its quality. The incident happened at a time when multiple high-ranking members of the Venezuelan government were being investigated for their involvement of drug trafficking.
On 18 November 2016, Flores' two nephews were found guilty of trying to ship drugs into the United States so they could "obtain a large amount of cash to help their family stay in power".