Human skin can defend itself against potentially invading microorganisms by production of antimicrobial peptides (AMPs). The expression of AMPs in atopic dermatitis (AD) is still emerging. To gain more insight into the role of AMPs in AD, we systematically analyzed the expression of ribonuclease 7 (RNase 7), psoriasin, and human β-defensins (hBD)-2 and -3 in AD compared with psoriatic and healthy control skin as well as after experimental barrier disruption. Immunostaining revealed enhanced expression of all AMPs in the lesional skin of untreated AD and psoriasis when compared with non-lesional skin and controls. Accordingly, induced in vivo secretion of RNase 7, psoriasin, and hBD-2 was detected using ELISA on lesional skin in AD and in even higher concentrations in psoriasis. The secretion of AMPs did not correlate with severity of AD and Staphylococcus aureus colonization. Skin barrier disruption caused enhanced immunoreactivity of hBD-2 and hBD-3 after 24 hours. Strong secretion of RNase 7 was already detected after 1 hour, whereas hBD-2 secretion was significantly enhanced after 24 hours only under occlusion. Thus, a disturbed skin barrier may trigger AMP induction in AD and psoriasis. The functional role of AMP in AD, especially with regard to the control of S. aureus colonization, needs further analysis.