The human beta-defensins (hBDs) are peptides with a strong antimicrobial activity. Patients with atopic dermatitis (AD) and mycosis fungoides (MF) are prone to skin infections. We aimed to investigate the mRNA expression of hBDs in lesional and non-lesional skin of MF patients, and to compare the data with hBD levels found in AD patients and healthy controls. In this prospective pilot study, 13 patients with MF were recruited. Punch biopsies were harvested from the centre of the tumour (lesional) as well as a healthy skin site (non-lesional controls). In addition to the specimens of MF patients, skin samples (healthy controls) were obtained from healthy subjects (n = 15) and patients with acute AD (n = 14). In order to detect mRNA of hBDs, we performed quantitative real-time reverse transcriptase polymerase chain reaction. As compared to healthy controls, skin of patients with MF (non-lesional and lesional) and AD patients showed significantly lower hBD-1 mRNA expression and significantly higher hBD-2 and hBD-3 mRNA expression. HBD-1 mRNA levels of lesional skin were significantly lower than those of non-lesional skin. By contrast, significantly increased hBD-2 and hBD-3 mRNA expression was found in lesional skin of MF patients when compared to non-lesional skin. HBD mRNA expression in lesional skin of MF patients did not significantly differ from hBD expression that was observed in AD lesions. We observed an identical pattern of hBD expression in MD and AD suggesting a common regulatory mechanism that might mainly be driven by T helper 2 lymphocytes.