Yem Chrin, 60, was arrested in 2014 in Battambang province after most of his patients had their blood tested positive for HIV and accused him of transmitting the virus via the reuse of unsterilized needles, Xinhua new agency reported on Saturday.
In December 2015, the Battambang Provincial Court found him guilty of committing "cruel torture" and decided to sentence him to 25 years in prison.
The court also ordered him to pay between $500 and $3,000 in compensation to each of more than 100 victims, who filed the complaints.
In September 2017, the Appeal Court decided to uphold the Battambang Provincial Court's ruling against Chrin.
"The Supreme Court sees that the Appeal Court's decision against Yem Chrin is correct, so the court decides to uphold the decision," Supreme Court Judge Nil Non said as he pronounced the verdict on Friday.
During a hearing last month, Chrin, who had been a village medical practitioner for almost 20 years, acknowledged his mistake and pleaded the court to reduce his jail sentence to 10 years.
He told the court that he reused syringes on multiple patients because it was difficult to get new ones.
The HIV outbreak in Battambang province's rural Roka commune, which came to light in 2014, had left some 290 people infected.
Cambodia currently has approximately 70,000 people living with HIV/AIDS and about 60,630 of them have received anti-retroviral drugs, according to the National AIDS Authority.
( With inputs from IANS )