ObjetiveTo evaluate the effectiveness of vagino-rectal swab autotomy for prenatal screening for GBS infection and to identify the barriers and facilitators encountered by the pregnant woman for this intervention. DesignCross-sectional study of diagnostic tests. Participants and siteA total of 213 pregnant women who attended the primary care midwife's office in 6 health centers of the Basque Health Service/Osakidetza in Bizkaia, who met the inclusion criteria and agreed to participate in the study, participated in the study. Main measurementsThe result of the vagino-rectal culture obtained by the pregnant woman was compared with the result of the vagino-rectal culture taken by the midwife in consultation on the same day, and the barriers and facilitators encountered by the women in the self-test were collected. ResultsSelf-testing as a test for GBS was found to have a sensitivity of 93.3% (95% CI 78.7–98.2), a specificity of 99.4% (95% CI 96.5–99.9), a positive predictive value of 96% (95% CI 82.8–99.4) and a negative predictive value of 98.8% (95% CI 95.6–99.7). 27.3% of respondents encountered some difficulty in the collection, only 4.8% did not feel qualified, 84.2% felt comfortable, 99.5% considered the information provided to be adequate and complete, 94.7% did not find the steps to follow complicated, and 96% were satisfied with the study. ConclusionsSelf-collection of vagino-rectal exudate for GBS detection has proved to be valid and reliable, which would make it possible to offer this option to pregnant women in the systematic screening for GBS infection.