The "vast majority" of those who tested positive had not shown any symptoms of COVID-19, the company said.
Operations have been halted at the world's deepest operational mine after more than one-quarter of COVID-19 tests given to those who work there produced positive results.
In terms of level 3 lockdown, underground mines are permitted to ramp up to 100% of production whereas they are now restricted to 50% of productive capacity under level 4 lockdown rules.
According to AngloGold Ashanti, it had conducted 650 tests at Mponeng since detecting the first case of the virus there last week, and 164 employees had tested positive with only a handful of tests left to process.
It added that most of the cases were asymptomatic and all those who tested positive would be isolated in line with national health protocols.
The mine, which had been operating at 50% capacity with a workforce of 2,400, has temporarily halted operations.
"The Gauteng department of health can confirm that 53 employees at AngloGold Ashanti Mponeng mine have tested positive to COVID-19".
In a national address, South African President Cyril Ramaphosa that his country will further ease its strict nationwide lockdown imposed several weeks ago to curb the spread of coronavirus disease 2019 (Covid-19), allowing most of the economy to function starting June 1.
Mining is a critical element in South Africa's already struggling economy, contributing eight percent to overall gross domestic product and raking in 45 percent of foreign exchange earnings.
AngloGold Ashanti's Mponeng Mine in Carletonville. Mines owned by Impala Platinum and Assore have closed temporarily due to the same issue.
South Africa president Cyril Ramaphosa had warned earlier this month there would be an "inevitable increase" in the number of cases as more people returned to work.
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