Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's dermal layers, causing significant discomfort, foul-smelling discharge, disfigurement, contractures, and scarring, which severely diminish the quality of life. HS is associated with alterations in the skin microbiome, impacting immune regulation and the skin's defense against harmful bacteria. Despite its prevalence, the contribution of the HS microbiome to disease pathology and the limited response to treatment remains largely unknown. To date, multiple 16S rRNA sequencing studies on HS tissue have only achieved genus-level granularity, identifying an increase in Gram-negative anaerobes and a decrease in skin commensals. A deeper understanding of microbial dysbiosis in individuals with HS is essential for optimizing treatment strategies. This requires a two-pronged approach to assessing the HS microbiome, including the isolation of bacterial species, which are often underutilized in translational studies focused on skin disorders. Isolating individual microorganisms from HS tissue is crucial for elucidating the role of bacteria in HS pathogenesis. Here, we highlight reproducible methods to successfully isolate anaerobic pathogens from HS tunnel tissue, providing the initial and most critical step in understanding bacterial role in HS. This method paves the way for targeted research into microbial contributions to HS and for developing more effective, personalized treatment strategies that address the complex microbial burden of this chronic condition.