Professional hunter Theo Bronkhorst and farm owner Honest Ndlovu, were charged with poaching offences and for not having the required hunting permit.
The pair were granted bail of $1,000 each (Â£638) and ordered to appear in court again on 5 August.
Walter Palmer, the US dentist who shot the animal known as Cecil, has left Zimbabwe but could also face charges.
Mr Palmer said he paid for the hunt, but was not aware of the lion's identity.
He said he regretted shooting the animal and believed he was on a legal hunt. He relied on professional guides to find a lion and obtain the necessary permits, he said.
Mr Bronkhorst and Mr Ndlovu could face up to 15 years in prison if found guilty.
Cecil is believed to have died on 1 July, but the carcass was not discovered until a few days later.
Mr Palmer is said to have shot and injured the animal with a bow. The group did not find the wounded lion until 40 hours later, when he was shot dead with a gun.
Separately, court records have shown that Mr Palmer has a felony record in the US after killing a black bear in the state of Wisconsin in 2006.
The dentist was given a one-year probation and fined $3,000, having shot the creature outside an authorised zone and then tried to pass it off as having been killed elsewhere.
Records from the Minnesota Board of Dentistry also show that Mr Palmer was the subject of a sexual harassment complaint which was settled in 2006.
A receptionist alleged that he had made indecent comments to her. Mr Palmer admitted no wrongdoing and agreed to pay out more than $127,000.
The American tourist is believed to have paid about $50,000 to go on the hunt in Zimbabwe.
More than 265,000 people have signed an online "Justice for Cecil" petition, calling on Zimbabwe's government to stop issuing hunting permits for endangered animals.
As news of the killing and details about the perpetrator spread online, there was a slew of comments on social media condemning Walter Palmer, with some people calling for him to face justice.
How the internet descended on the man who killed Cecil the lion
Mr Palmer insists that he believed his guides had secured "all proper permits" for the hunt.
"I relied on the expertise of my local professional guides to ensure a legal hunt," he said in a statement on Tuesday.
"I deeply regret that my pursuit of an activity I love and practice responsibly and legally resulted in the taking of this lion."
He said he had not been contacted by authorities in Zimbabwe or the US but would "assist them in any inquiries they may have".
The dentist is believed to be back in the US, although his exact whereabouts are unknown.
His dental practice was closed on Tuesday and a note was placed on the door referring visitors to a public relations firm.
Cecil the lion was skinned and beheaded, according to the Zimbabwe Conservation Task Force (ZCTF), a local charity.
The ZCTF said the hunters had used bait to lure him outside Hwange National Park during a night-time pursuit.
35,000
Max estimated lion population
12,000
Max lion population in southern Africa
665 Approx number of 'trophy' lions killed for export from Africa per year
49 Lion 'trophies'  exported from Zimbabwe in 2013
0.29% Contribution to GDP of Zimbabwe from trophy hunting
17% Of Zimbabwe's land given  to trophy hunting
The animal had a GPS collar fitted for a research project by UK-based Oxford University that allowed authorities to track its movements. The hunters tried to destroy it, but failed, according to the ZCTF.
On Monday, the head of the ZCTF told the BBC that Cecil "never bothered anybody" and was "one of the most beautiful animals to look at".
Two men accused of helping a US tourist hunt and kill Zimbabwe's most famous lion have been released on bail.