Sexually transmitted infections (STIs) constitute an important world-wide public health problem. The use of sensitive and specific laboratory methods for diagnosing this condition is crucial to reduce the transmission and sequelae of STI. The present review describes current microbiological methods for the diagnosis of STIs. Neisseria gonorrhoeae and Chlamydia trachomatis are the pathogens most frequently involved in urethral and cervical infection. Culture continues to be the gold standard for diagnosing gonorrhea. Nucleic acid amplification assays are considered the new gold standard for C. trachomatis, although culture is till the most specific technique. Genital ulcers due to Treponema pallidum, Haemophilus ducreyi, or herpes simplex virus have little clinical and bacteriological correlation; therefore, it is essential to establish the microbiological diagnosis. Lesions present in the primary or secondary period of syphilis can be diagnosed by dark field microscopy. Serologic diagnosis for the remaining periods is based on non-treponemal tests associated with confirmatory treponemal tests. Cell culture is considered the gold standard for herpes simplex virus although molecular methods also have a sensitivity and specificity near 100%. Currently, microbiologic diagnosis of H. ducreyi and venereal lymphogranuloma is achieved with the use of molecular methods on samples obtained from the ulceration or lymph adenopathy. The diagnosis of genital warts in immunocompetent patients is based on clinical findings in most cases because the lesions are sufficiently characteristic. Culture is considered the reference method in Trichomonas vaginalis infection.