Background: Microbiome-targeted treatments have been investigated in atopic dermatitis (AD). We aimed to investigate the use of probiotic Lactococcus lactis lysate cream in AD. Methods: 13 patients with mild-to-moderate AD were treated with differently concentrated probiotic creams (3%, 10% and 30%) or placebo cream for 4 weeks. Disease severity (EASI, IGA), epidermal barrier function (TEWL) and patient-reported impact (DLQI, POEM, ADCT, pruritus and sleep disturbance VAS) were measured at baseline, 4 and 8 weeks. Comprehensive clinical data and laboratory values (blood eosinophil count, total serum IgE-levels and specific IgEs to aeroallergens) were obtained. Results: Comparison of the treatment groups showed no clear differences regarding AD severity (EASI, p=0.76, CI: 0.65-1.00), epidermal barrier dysfunction (TEWL, p=0.37, CI: 0.19-0.73) or patient-reported impact (DLQI, p=0.76, CI: 0.65-1.00; POEM, p=0.76, CI: 0.35-0.88; ADCT, p=0.72, CI: 0.65-1.00; pruritus VAS 0.67, CI: 0.55-1.00; sleep disturbance VAS, p=1.00, CI: 0.79-1.00) between different probiotic lysate concentrations and placebo. The probiotic lysate cream was well tolerated and there were no significant adverse effects. Limitations were a small and heterogenous patient groups and a relatively short follow-up with no evaluation of long-term effects. Conclusions: Topical probiotic L. lactis lysate cream showed no clear differences between the tratment groups in mild-to-moderate AD. Although topical probiotics have been reported effective in a limited number of studies, more placebo-controlled clinical studies are needed to explore their potential role in the treatment of AD.