Non-viral sexually transmitted infections (STI) are an important cause of physical, psychological and social distress, have severe consequences for women's reproductive health and may be transmitted to the newborn child. These infections are also risk factors for the acquisition and transmission of HIV and other STI, and for premature labor. In the last years we have observed a gradual decrease in the national incidence of gonorrhea. The implementation of a screening program in our country for Chlamydia trachomatis is necessary, since up to 80% of infections in women are asymptomatic. Due to medical, psychosocial and legal reasons, laboratory diagnosis of STI has to be certain. This offers a great challenge to laboratories. Since etiological agents are susceptible to environmental conditions, present a high adaptation to their human host and have particular physiological characteristics, their laboratory diagnosis is more difficult than diagnosis of conventional microorganisms. Otherwise, the diagnostic techniques currently available for non-viral STI are characterized by their excellent sensitivity and specificity, which result of great interest given the curable nature of these infections. Clinical specimens obtained for diagnosis of STI and other genital infections, such as bacterial vaginosis or Candidiasis represent a large proportion of specimens processed by clinical laboratories. Thus, the creation of norms and quality control guidelines for laboratories which diagnose these infections, and also the epidemiologic and genetic surveillance of circulating sex transmitted microorganisms should be considered a priority in our country. The objective of this study is to review current literature on accurate diagnostic procedures especially for three non-viral STI agents: C. trachomatis, N. gonorrhoeae, and Trichomonas vaginalis.