Self-described "knitting nannas" Helen Kvelde and Dominique Jacobs are challenging the laws, with the backing of the Environmental Defenders Office (EDO).
"We need to defend our freedom to protest," Ms Kvelde told reporters on Thursday.
"Once it has been eroded it is gone forever," she said.
Ms Jacobs called the laws a "slap in the face".
"Australians shouldn't have to risk imprisonment or bankruptcy to participate in our democracy," she said.
EDO chief executive David Morris said the laws that passed in April were too broad, vague and could be used to suppress protests.
"These types of laws, they erode the fabric of our democracy," he told reporters.
"In passing these laws they have really placed some great restraints on the way in which people might choose to peacefully protest, added to that they increased the penalties," Mr Morris said.
Amendments to roads and crimes legislation set penalties of up to two years' jail and fines up to $22,000.
New offences targeted people blocking access to major facilities, but critics argue the law is too broad.
The use of the word "near" and a broad definition of "major facility" that includes ports, railways, train stations, and any infrastructure facility providing a service to the public is the main point of concern.
Western Sydney University Associate Professor Azadeh Dastyari says states are allowed to protect the public and the peace.
"But it has to be proportionate," she said.
"Inconvenience is not a lawful reason to crush protests."
There were concerns the vague and broad wording of the legislation meant it could be used against other protesters on a whim.
The government said that was not the bill's intention, but rejected amendments seeking to ensure people do not need permission from the police or the state to protest.
"It's so vague and broad that we actually don't know exactly how it can be used," Prof Dastyari said.
The laws came after Blockade Australia climate activists staged a series of high-profile protests blocking traffic on major roads and preventing access to Port Botany in Sydney's south.
Attorney-General Mark Speakman called the Greens who opposed the changes "anarchists".
Labor supported the government legislation with Opposition Leader Chris Minns saying blockading protesters were alienating people from their cause.
Independent Sydney MP Alex Greenwich told parliament the laws showed MPs at their worst and blaming sections of the media for stoking an unnecessary sense of urgency.
"The government and the opposition have met the deadline that was set by (a Sydney commercial radio station) to rush through this poorly drafted legislation, which will have no meaningful impact," he said.
The civil case in the Supreme Court challenging the laws is set for a directions hearing on October 26.
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