The objective of the study was to determine the frequency of microbial invasion of the amniotic cavity in the midtrimester of pregnancy in patients undergoing amniocentesis for clinical indications. This was a prospective investigation of the amniotic fluid of 344 asymptomatic women recruited in midpregnancy for the presence of microbial DNA. Amniotic samples obtained at the time of amniocentesis for genetic testing on women between 15 and 22 weeks of gestation were tested specifically for the presence of Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium as well as for other bacteria and fungi using broad-range polymerase chain reaction only. Pregnancy outcomes were reviewed independent of all molecular test results. Using broad-range polymerase chain reaction, the prevalence of microbial invasion of the amniotic cavity in women between 15 and 22 weeks of gestation was 0% (0 vs 344). Early preterm delivery occurred in only 4 women (1%); 1 delivered electively and 3 spontaneously. None were associated with Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, or Mycoplasma genitalium. In addition, broad range polymerase chain reaction did not reveal the presence of other bacterial or fungal microbes. Microbial invasion of the amniotic cavity in midtrimester gestations of low-risk pregnant women was not detected using molecular methods in 344 patients.