<b><i>Background:</i></b> Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory skin disease involving the follicular unit. Growing evidence suggests a crucial role of hyperkeratosis, infundibular plugging and perifolliculitis in its pathogenesis. <b><i>Objectives:</i></b> To characterize the microscopic morphology of open comedos using in vivo reflectance confocal microscopy (RCM) and define alterations occurring in HS normal-appearing perilesional skin compared to healthy skin of a control group, discussing therefore microscopic differences. <b><i>Materials and Methods:</i></b> Twenty patients (11 males, 9 females; aged 19–51 years) affected by HS were recruited. RCM was performed on open comedos of axillary/inguinal regions and on normal-appearing skin areas distant at least 1.5 cm from HS lesions. Ten healthy individuals (6 males, 4 females) were included as control group. <b><i>Results:</i></b> RCM aspects of 78 open comedos were explored, observing circular/ovoidal structures disclosing a hyperrefractive amorphous material (72/78, 92.3%) within the infundibular cavity surrounded with a bright (51/78, 65.4%) and regular (52/78, 66.2%) border. Follicular ostia of HS perilesional skin (<i>n</i> = 541) compared to follicular ostia of a control group (<i>n</i> = 321) were characterized by a larger infundibular diameter (183.4 vs. 85.8 µm) and diverged in terms of material collected inside infundibula (44.5 vs. 21%) and keratinization of the border (47.6 vs. 25.5%). <b><i>Conclusion:</i></b> In vivo characterization of open comedos and the recognition of microscopic subclinical alterations of HS normal-appearing skin, compared to healthy skin, could add further insights into the definition of biological events leading to HS manifestations, including the dysregulated process of keratinization.