The staff from the Zoe's Ark charity held hands as they arrived at court. They say the children were orphans from Darfur who were being taken to France.
Their leader testified the operation had the support of the French and Chadian governments and the UN.
The six, who have gone on hunger strike, could face 20-year hard labour terms if found guilty.
The French government has appealed for leniency.
The court will try to establish whether the children were the beneficiaries of a humanitarian project that went wrong, or the victims of child-trafficking.
'Truth'
The BBC's John James in N'Djamena says the leader of the six, Eric Breteau, looked gaunt but composed in court.
"I dispute the charges I am accused of. I intend to respond point by point," he told the packed courtroom.
"Nobody ever expressed even the tiniest doubt about the Sudanese origins of the children present at our bases," Mr Breteau told the court, apparently in response to accusations that the children were not really from Darfur. 
He said the operation had been kept secret from local authorities in Chad "in order not to put the mission in danger ... and so as not to alert the suspicions of the Sudanese secret service," Mr Breteau said, quoted by the AFP news agency.
"Before the operation was launched... all the French authorities were informed in writing of our action," he added.
The governments of France and Chad have denied any knowledge of the operation.
The accused chatted to each other during breaks in proceedings but otherwise followed closely.
The court was surrounded by anti-riot police, armed with machine-guns.
The courtroom was packed, included many journalists from the French media. Outside, there was a crowd of about 100 people.
The case has sparked protests in oil-producing Chad amid claims the accused might benefit from special treatment because they are Europeans.
"Our clients will tell the truth, that they came here to do good," said Mario Stasi, lawyer for the charity's nurse, Nadia Merimi.
The six were stopped by officials as they went to catch a flight from Chad to France in late October with the children.
Most of the children have been found to have at least one living parent or guardian.
'Fight to the end'
The judge will want to know why, when none of the children was injured, they were bandaged and caked in fake blood ahead of their flight.
One of the accused, Emilie Lelouche, told France Info radio on Thursday: "We're going to fight to the end."
The aid workers have been on a hunger strike since earlier this month, refusing food but drinking water, and have accused the French government of deserting them. 
On Thursday, French Junior Foreign Minister Rama Yade said the government had appealed to Chad to treat the accused with a "certain clemency".
French Justice Minister Rachida Dati said this week that if convicted the six might be able to serve their sentences in France because of a bilateral judicial agreement.
Analysts have speculated a diplomatic deal could be struck after French President Nicolas Sarkozy met Chadian President Idriss Deby last week in Lisbon during a European and African leaders' summit.
But an unnamed Chad government official told Reuters news agency: "Have you ever seen an African sent back home after committing a crime as serious as the one [allegedly] committed by the members of Zoe's Ark?"
Three Chadians and one Sudanese refugee are also on trial for conspiracy.
In all, 17 Europeans were arrested in the eastern Chadian town of Abeche on 25 October but 11 others - three French journalists, seven Spanish flight crew and a Belgian pilot - have since been repatriated.
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