Background Skin infections related to disrupted antimicrobial defence are a common problem in atopic dermatitis (AD). Altered levels of antimicrobial peptides, including human β-defensin (hBD)-2, have been reported in AD skin, and a link to impaired barrier function has been suggested. Objectives To study hBD-2 in relation to skin barrier function in patients with AD and controls, and to study hBD-2 in relation to disease severity. Methods Twenty-five patients with AD and 11 controls were enrolled. hBD-2 peptide concentration was determined in stratum corneum samples collected by a minimally invasive tape-stripping method. Disease severity was assessed by SCORing Atopic Dermatitis (SCORAD), and skin barrier function was evaluated by measurement of transepidermal water loss (TEWL) and skin pH. Patients with AD were characterized according to filaggrin mutations. Results hBD-2 concentrations in the stratum corneum were found to differ between lesional and nonlesional AD skin and controls, with the highest values in lesional skin (P < 0·001). SCORAD and TEWL were significantly increased in participants with measureable hBD-2 (P < 0·018 and P < 0·007, respectively). Significant correlations between hBD-2 in lesional skin, and TEWL and SCORAD were observed (R = 0·55 and R = 0·44, respectively). No correlations with skin pH were found. hBD-2 was not found to relate to filaggrin mutations. Conclusions A significant correlation was found between hBD-2, disturbed skin barrier function and disease severity. The minimally invasive skin sample technique enables evaluation of the stratum corneum and its proteins over time and provides the possibility of relating these findings to treatment, infections and physiological variations. What's already known about this topic? Human β-defensin (hBD)-2 is expressed in lower quantities in skin biopsies from patients with atopic dermatitis (AD) than in patients with psoriasis. hBD-2 levels are higher in AD lesional skin than in nonlesional skin. What does this study add? A new minimally invasive skin sample technique was used. A correlation between hBD-2 and disease severity was found, along with a correlation with barrier impairment as measured by transepidermal water loss. hBD-2 was not found to relate to filaggrin mutations or skin pH.