Background: The irritant sodium lauryl sulfate (SLS) is known to cause a decrease in the stratum corneum level of natural moisturizing factor (NMF), which in itself is associated with changes in corneocyte surface topography. Objective: To explore this phenomenon in allergic contact dermatitis. Methods: Patch testing was performed on patients with previously positive patch test reactions to potassium dichromate (Cr), nickel sulfate (Ni), methylchloroisothiazolinone (MCI)/methylisothiazolinone (MI), or p-phenylenediamine. Moreover, a control (pet.) patch and an irritant (SLS) patch were applied. After 3 days, the stratum corneum from tested sites was collected, and NMF levels and corneocyte morphology, expressed as the amount of circular nanosize objects, quantified according to the Dermal Texture Index (DTI), were determined. Results: Among allergens, only MCI/MI reduced NMF levels significantly, as did SLS. Furthermore, only MCI/MI caused remarkable changes at the microscopic level; the corneocytes were hexagonal-shaped with pronounced cell borders and a smoother surface. The DTI was increased after SLS exposure but not after allergen exposure. Conclusions: MCI/MI significantly decreased NMF levels, similarly to SLS. The altered corneocyte morphology suggests that skin barrier damage plays a role in the pathogenesis of MCI/MI contact allergy. The DTI seems to differentiate reactions to SLS from those to the allergens tested, as SLS was the only agent that caused a DTI increase.