Introduction: Increasing usage of antimicrobial agents may contribute to bacterial resistance in atopic dermatitis (AD). In this case an alternative topical treatment might be gentian violet (GV), suggested for its antibacterial and antifungal properties. Aim: To assess the microbial composition of lesional skin in children with AD and a control group aged 2-12 years, before and after 3 days of 2% aqueous GV application. Material and methods: Skin samples were taken from 30 AD patients and 30 healthy controls aged 2-12 years. The procedure was done two times -before and after 3 days of 2% aqueous GV application. The material was collected from skin lesions in the cubital fossa using 25 cm 2 impression plates, containing CHROMagar Staph aureus and CHROMagar Malassezia. After the incubation period, the grown colonies were counted and identified by the Phoenix BD testing system. Results: The results revealed a statistically significant reduction in total counts of bacteria in both groups of children after GV application (p < 0.05). The significant decrease in the number was seen in Staphylococcus spp. (S. aureus, S. capitis, S. haemolyticus, S. cohnii) in AD patients. The number of Staphylococcus spp. was comparable in patients with AD after GV treatment and healthy patients before GV exposure (p = 1.000). Conclusions: Our study results show that GV does not damage the skin surface ecosystem and allows the reduction of excessive bacterial counts on eczematous lesions to a 'safe' level, similar to that of healthy children.