Bovine digital dermatitis (DD) is the most relevant infectious disease associated with lameness affecting cattle worldwide. Notwithstanding decades of research, the pathogenesis of this disease and the potential reservoir of pathogenic microbes involved in DD remain unclear. Here, we characterize the microbiome of healthy skin (HS) and lesions from dairy cows affected with different stages of DD and we also identified DD-causing Treponema spp. in rumen and fecal samples. A total of 140 biopsy samples (BS) (51 HS and 89 DD lesions) were collected from Holstein dairy cows housed in 3 different dairy farms. BS were trimmed in 2 different layers, resulting in 280 samples. Rumen fluid (n = 8) and fecal (n = 14) samples were also collected. DNA was extracted and the microbiome was determined by shotgun and 16S metagenomic techniques using Illumina MiSeq platform. Discriminant analysis revealed that microbiomes of HS, active and inactive DD lesions were completely distinct. The differences in microbiomes between the superficial and deep strata were found to be minor. Treponema spp. were found in greater (P < 0.05) relative abundance in active DD lesions when compared with HS and inactive DD lesions and these Treponema spp. were nearly ubiquitously present in rumen and fecal microbiomes. Candidatus amoebophilus asiaticus, a bacterium not previously reported in DD lesions, was encountered in high (P < 0.05) relative abundance in active and inactive lesions but not in HS. In conclusion, our data support the concept that DD is a polymicrobial disease, with active DD lesions having a markedly distinct microbiome dominated by T. denticola, T. maltophilum, T. medium, T. putidum, T. phagedenis and T. paraluiscuniculi. Furthermore, these Treponema species are nearly ubiquitously found in rumen and fecal microbiomes, suggesting that the gut is an important reservoir of microbes involved in DD pathogenesis. Further investigation into the potential role of the gut microbiome as a reservoir for pathogens leading to DD development and of prophylactic measures to control the potential environmental shedding of these pathogens is needed.