Morgan Geyser and Anissa Weier have been charged with attempted murder over the attack.
Last week, a Wisconsin judge decided that they should be tried as adults, meaning they could face decades in jail if convicted.
The victim, 12 at the time, was stabbed 19 times but survived.
She was found by a cyclist crawling from the woods where she was attacked with stab wounds to her arms, legs and torso, after the attack in May 2014.
A judge entered not guilty pleas on behalf of both girls at a hearing on Friday.
Investigators say the two girls had been plotting for months to kill their victim in "dedication" to Slender Man, a fictional horror website character.
They spoke of their desire to become the paranormal figure's "proxies" by killing to demonstrate their loyalty, police said.
A lawyer for one of the girls told the AP news agency before the hearing that his client would enter a plea of not guilty by reason of mental disease or defect.
Their lawyers have previously argued for the case to be heard in juvenile court, saying both teenagers were suffering from mental illness.
Following the attack, the suspects were found walking near a local highway and a knife was found in one of their backpacks.
The victim has recovered and since returned to school.

Cambridgeshire and Peterborough Combined Authority (CPCC), set up this year, originally estimated annual running costs of about £1.3m.
But the authority's scrutiny committee voted against approving it, with one member asking if recruitment plans were "written on the back of a bus ticket".
CPCC said a reason for the rise was that a new authority was being created.
The authority, created under government devolution plans, intends to employ 15 staff, including newly-created housing, skills and transport director roles all on annual salaries above £100,000.
After Wednesday's decision by the scrutiny committee, the process of recruiting any new staff cannot begin until CPCC has reconsidered its budget, the deadline for which is 31 August.
Labour Cambridge city councillor Dave Baigent, a member of the scrutiny committee, asked of the new jobs: "Were these ideas written down on the back of a bus ticket? Were they discussed with councillors in advance?"
Lucy Nethsinga, a Liberal Democrat county councillor, said she had concerns over how these jobs overlapped with those in county and district councils.
CPCC chief executive Martin Whiteley said: "We, with 15 staff, will be substantially smaller than any other devolved authority in the country.
"I don't envisage us creating a big shiny office. We'll be looking to work flexibly across council offices."

Welsh language minister Alun Davies told AMs it would help efforts to reach that goal stay on the right track.
Targets to meet growing demand for Welsh-speaking teachers and public sector workers will also be set.
Culture committee chairwoman Bethan Jenkins said AMs had been told 70% more Welsh-medium teachers were needed.
Mr Davies responded that around a third of teachers in Wales could speak Welsh, and that the challenge was to see if more of them would be willing to teach through the medium of Welsh.
Earlier this month, Welsh language commissioner Meri Huws called for "radical change" in the education system to ensure all children under the age of seven were "immersed" in Welsh.
Two 13-year-old girls accused of stabbing a classmate to please the online horror character Slender Man have pleaded not guilty in court.
[NEXT_CONCEPT]
A new devolved authority has been ordered to review its estimated budget after it rose by £500,000.
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The Welsh Government will set a "framework" of five-year targets in its attempt to ensure a million people speak Welsh by 2050.