The pop diva arrived in South Korea on Friday, a week before her Seoul performance which kicks off her Born This Way Ball Global Tour.
About 300 Protestant church members gathered in downtown Seoul to hold a group prayer against the concert in the capital, Kang Ju-hyun, a prayer organiser said.
"We will pray to God that the concert will not be realised so that homosexuality and pornography will not spread around the country," he said.
Kang's group last week put out street banners in Seoul accusing the eccentric singer of "spreading unhealthy sexual culture" through "lewd lyrics and performances," before they were removed by the city officials.
South Koreans aged under 18 have been banned from the much-anticipated concert after it was rated unsuitable for younger audiences.
The show had an initial age rating of 12 and older but the Korea Media Rating Board — a state watchdog — adjusted it upwards.
