Background: NICU patients are at risk of skin breakdown due to prematurity, irritant exposure, medical status and stress. There is a need to minimize damage, facilitate skin development and reduce infection risk, but the literature on the effects of skin care practices in NICU patients is limited. Objectives: To test the hypothesis that baby diaper wipes with emollient cleansers and a soft cloth would minimize skin compromise relative to cloth and water. Methods: In 130 NICU infants (gestational age 23–41 weeks, at enrollment 30–51 weeks), measurements of skin condition, i.e., skin erythema, skin rash, transepidermal water loss (TEWL) and surface acidity (pH), within the diaper and at diaper and chest control sites were determined daily for 5–14 days using standardized methods. Treatments were randomly assigned based on gestational age and starting skin irritation score: wipe A, wipe B, and the current cloth and water NICU standard of care. Results: Perineal erythema and TEWL were significantly lower for wipes A and B than cloth and water beginning at day 5 for erythema (scores of 1.11 ± 0.05, 1.2 ± 0.05, and 1.4 ± 0.06, respectively) and day 7 for TEWL (28.2 ± 1.6, 28.8 ± 1.6, and 35.2 ± 1.6 g/m2/h, respectively). Wipe B produced a significantly lower skin pH (day 5, 5.47 ± 0.03) than wipe A (5.71 ± 0.03) and cloth and water (5.67 ± 0.04). The starting skin condition, stool total, age and time on current standard impacted the outcomes. Conclusions: Both wipes are appropriate for use on medically stable NICU patients, including both full and preterm infants, and provide more normalized skin condition and barrier function versus the cloth and water standard. Wipe B may facilitate acid mantle development and assist in colonization, infection control and barrier repair. Neonatal skin continues to change for up to 8 weeks postnatally, presumably as it adapts to the dry extra-uterine environment.