The charges are the culmination of a 16-month investigation into the administration of dubious supplements in 2012 at the Melbourne-based Essendon Bombers, one of the Australian Football League's oldest and most powerful teams.
"The Australian Sports Anti-Doping Authority has put formal allegations of possible anti-doping rule violations to 34 current and former players from the Australian Football League," the agency said.
"Based on the advice of our legal counsel and a review of the evidence... I have reached the conclusion that these players have a case to answer under the World Anti-Doping Code," ASADA CEO Ben McDevitt said.
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The AFL disqualified Essendon from the competition's play-offs last year, banned the club's head coach James Hird for 12 months and slapped the team with a record fine for bringing the full-contact indigenous game into disrepute.
The punishments followed an independent probe commissioned by the club which found governance failures had contributed to a "disturbing picture of a pharmacologically experimental environment".
ASADA is also investigating the administration of banned supplements to multiple players at one of the country's top-flight rugby league teams, the Sydney-based Cronulla Sharks, and is expected to lay charges in coming days.
Stephen Dank, a sports scientist formerly employed by Essendon and who also worked with the Sharks, was charged by ASADA in March for his part in the supplements programme at Essendon but has yet to face further action.
He has consistently denied wrongdoing and said he would fight the charges to the highest court.
The scandal, which has overshadowed two AFL seasons, has unearthed grisly details about a club's determination to seek an edge in the country's premier sports competition.
Local newspapers have reported grisly details of players being taken away from their training base to be injected with peptides in private clinics.
Peptides are short chains of amino acids which athletes can take in supplement form to aid muscle growth and re-generation.
A number of them are banned by the World Anti-Doping Agency (WADA), including growth hormone and Insulin-like Growth Factor-1 (IGF-1).
The scandal threatens to bring Essendon to its knees, with a majority of those charged still active players on the club's 47-man roster.
First-time doping offenders face a two-year ban, according to the WADA code, but could have their suspensions reduced if they cooperate with authorities.
The players have 10 days to respond to the charges, which ASADA refer to as 'show cause notices'.
"This information along with the evidence collected by ASADA will be put to the independent Anti-Doping Rule Violation Panel for consideration," ASADA said.
ASADA chief McDevitt said he hoped to cut deals.
"You could end up going from two years to 12 months to six months," McDevitt told state radio ABC.
"What I'm encouraging individuals now, and the club, and the AFL, is to have a think about this very carefully and have discussions with us."
Even six-month bans for all players involved would gut Essendon's playing roster, with the impact on sponsorship and season tickets certain to bite hard.
The club lies ninth in the 18-team table, halfway through the AFL's regular season.
Essendon declined to comment but issued a statement that chairman Paul Little would face the media later on Friday.
New AFL chief executive Gillon McLachlan, who has walked into a firestorm after replacing long-serving boss Andrew Demetriou last week, said the charges were "just the start of a long process".
"It's essentially a proposition, an allegation to which they have to respond," he told ABC radio. "It's not an indication of guilt."
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