White House spokeswoman Sarah Huckabee Sanders called the arrests "unjust" and said they follow Maduro's "outrageous seizure of power through a sham election" - a reference to Sunday's vote for an assembly to rewrite the constitution, which is opposed by 70 percent of Venezuelans.
The delegates had been expected to take their seats Thursday, but in a surprise move Wednesday evening, Maduro moved the start to 11 am on Friday - in the same building where the current opposition dominated National Assembly sits.
Independent analysts and opposition leaders had already contended that the actual participation level was much lower.
The size of the turnout has been central to the legitimacy of the vote, as both the government and the opposition argue that the other side does not have widespread support.
"Yesterday's illegitimate elections confirm that Maduro is a dictator who disregards the will of the Venezuelan people", US Treasury Secretary Steven Mnuchin told reporters.
Maduro provided no evidence to support his claim, but his remarks were received with resounding applause from about 500 people elected to the assembly. In early July, the opposition held a symbolic referendum against the constituent assembly, and said it drew around 7.1 million voters.
The us president, Donald Trump held "personally responsible" for his venezuelan counterpart of the treatment of the two opponents, condemning "the actions of the dictatorship Maduro".
A National Elections Council official contacted by Reuters said there was nobody available to speak about the issue.
"Any manipulation, like Mr. Mugica suggests, does not take into consideration that Smartmatic is part of the safety of the totalization system", Lucena said.
But generating a high turnout was considered vital to proving public enthusiasm for the new Constituent Assembly, which gives the government effective control over all branches of government. Washington is also said to be considering restricting imports of crude oil from Venezuela, among the top US suppliers.
In a letter, he said that following the "unjustified arrests" of opposition leaders Antonio Ledezma and Leopoldo Lopez, he would like to consider "freezing assets and imposing travel ban to the European Union to the members of the Venezuelan government including its President Nicolas Maduro and its entourage".
"The Maduro administration is sending a terrifying message to all people in Venezuela: dissent will not be tolerated in any form", said Erika Guevara-Rosas, Americas Director at Amnesty International.
Even a software company involved in setting up voting systems announced the turnout figure had been altered by at least one million votes.
Smartmatic said that to ensure there is no tampering, its tabulation centers must be audited by members of all political parties.
The former bus driver and trade union leader accused Smartmatic of being influenced by the USA in a speech on Wednesday in front of the newly-elected constituent assembly in Caracas.
Luis Vicente Leon, president of Datanalisis, a Caracas-based polling agency, said Smartmatic's finding was, "without a doubt, the most devastating pronouncement yet for the credibility" of the nation's electoral council.
On Wednesday night, one Labour MP, Chris Williamson, attacked fresh USA government sanctions on Caracas and suggested that people protesting against the Maduro government had been put up to it by right-wing opposition. Top military figures have been given special status and are scattered throughout the government.