Manning, who spent seven years in military prisons for leaking US military and diplomatic secrets to WikiLeaks in 2010 and then two more months in an Alexandria, Virginia, jail this year on contempt of court charges, said she would not bow to the threat of another contempt sentence.
She accused the government of seeking to revive her original court martial case, saying prosecutors were unhappy over her 2017 pardon by president Barack Obama.
"No matter what happens today, whether I am placed in confinement or not, I'm not going to comply with this grand jury," she told journalists outside the Alexandria courthouse.
"The goal here is really to relitigate the court martial. They didn't like the outcome -- I got out."
Manning, 31, was called early this year to testify to a grand jury -- a panel investigating major crimes that operates in secrecy -- about her work with Assange and WikiLeaks nine years ago.
She said the government was abusing the grand jury process and refused to testify, saying she had answered all the questions years before anyway.
A judge found her in contempt and on March 8 she was jailed indefinitely.
She was released last week when the grand jury's mandate expired, and was called to testify before a new one on Thursday which she said was seeking answers to the same set of questions.
While she was not at liberty to discuss the specifics of the investigation, she indicated that it also was a probe of Assange and WikiLeaks' actions in 2010.
The US Justice Department has asked Britain the extradite Assange to stand trial in the United States for "conspiracy" in advising Manning on breaking into a restricted US government computer.
Assange, now committed to a British prison for a year and also facing an extradition effort from Sweden, asserts that he is a journalist with the right to publish purloined secrets.
Manning, a transgender woman whom supporters call a whistleblower, said the new grand jury case is meaningless since the Justice Department already unveiled its charges against Assange.
"The case doesn't make sense, it's very bananas," she said.
"Ultimately this is an attempt to place me back into confinement."