Male genital lichen sclerosus (MGLSc) is a chronic inflammatory scarring dermatosis associated with penile carcinoma. The prepuce is pivotal in its etiology. Other proposed etiological factors are the subject of dispute and include occluded urinary exposure, autoimmunity, immunodysregulation, and infectious agents.To determine whether the bacterial microbiota of the balanopreputial sac and urine are associated with MGLSc.Twenty uncircumcised patients with MGLSc and 20 healthy uncircumcised males were enrolled in a prospective case-control study. Balanopreputial swabs and urine specimens were subjected to 16S rRNA gene amplicon sequencing.Microbiota analysis indicated differences between the groups. In the balanopreputial sac, the median relative abundance of Finegoldia spp. was lower (9% [range 0-60%]) in MGLSc patients than in controls (28% [range 0-62%]). Conversely, the median relative abundance of Fusobacterium spp. was higher in MGLSc patients (4% [range 0-41%]) than in controls (0% [range 0-28%]). In the urine, the median relative abundance of Finegoldia spp. was comparable between groups, whereas that of Fusobacterium spp. was higher in MGLSc patients (0% [range 0-18%] vs. 0% [range 0-5%]). There was a strong association between the microbiota composition of the balanopreputial sac and urine in MGLSc.Dysbiosis could be involved in the etiopathogenesis of MGLSc. Further studies are required to confirm the association suggested herein and to determine its nature.