Many diagnostic methods for sexually transmitted infections (STIs) have been developed. Because various infection agents are associated with STIs, and because infected persons sometimes show no symptoms, the diagnosis of STIs using nucleic acid amplification tests(NAATs) has required not only simultaneous multi-targeting, but also sensitive detection. Here, we compare microarray and real-time PCR for the detection of three common STIs agents, Ureaplasma urealyticum, Mycoplasma genitalium, and Chlamydia trachomatis, using human urine samples. The detection results showed that microarray and real-time PCR technology are both effective tools for the detection of STI agents. In conclusion, real-time PCR detection offers more sensitivity and specificity than microarray, because of the quantitative method employed. But, microarray offers better performance, in terms of high-throughput and simultaneous multi-targeting.