The Arsenal defender sustained the problem during the Gunners' Women's Super League defeat to Manchester United on Wednesday night, staying down after winning possession back from Katie Zelem.
Williamson immediately signalled that her problem was a serious one, and she was helped to her feet by medical staff before gingerly walking off the field.
The nature of the 26-year-old's fall immediately sparked concern about a long-term layoff, and the Lionesses' worst fears have now been confirmed.
Arsenal released a medical update on Friday afternoon confirming that Williamson had suffered an ACL rupture - the latest in a long line of prominent female footballers to fall victim to such an injury.
Williamson will undergo an operation "in due course" and is poised to miss the rest of the domestic season and the upcoming World Cup, which begins in July.
"We can confirm that Leah Williamson suffered a ruptured anterior cruciate ligament in our Barclays Women's Super League match against Manchester United on Wednesday night," the update read.
"Leah was substituted in the first half of the game at Leigh Sports Village and underwent further assessment on Thursday to determine the extent of the injury. Leah will now begin a period of rehabilitation and is set for an extended spell on the sidelines.
"She will undergo surgery in due course. Everyone at Arsenal will be supporting Leah closely throughout the journey ahead and we would ask that her privacy is respected at this time."
Williamson ends the 2022-23 campaign having scored one goal and provided three assists in 17 matches for Arsenal, whose head coach Jonas Eidevall hit out at the Leigh Sports Village pitch after the defender's enforced withdrawal.
The England captain's lengthy layoff - which could last anywhere between six and nine months - is another damaging blow to Arsenal's hopes of competing for WSL and Champions League glory in the final few weeks of the season.
The Gunners already lost Beth Mead and Vivianne Miedema to ACL injuries earlier in the season, while captain Kim Little was ruled out for the rest of the season with a hamstring problem earlier this week.
Arsenal's loss to Man United leaves the Gunners six points behind the Red Devils - who have played a game more - in the WSL table, while Chelsea are two points better off than Eidevall's side with a game in hand.
Focus will soon turn to the semi-finals of the Champions League for Arsenal, who host Wolfsburg in Sunday's first leg before travelling to the Volkswagen Arena on May 1.
Chelsea duo Fran Kirby and Millie Bright are also World Cup injury doubts for England, who will face Haiti, Denmark and China in Group D in Australia.