The biodiversity hypothesis posits that contact with microbial biodiversity is beneficial for human health, and that the loss of such contact may contribute to immune-mediated diseases such as atopy. Based on this hypothesis, we aimed to investigate whether a lotion containing a highly diverse mixture of inactivated natural microbes is a safe and feasible method to administer nature-based microbial exposure to humans, and to assess its effects on healthy and atopic skin. Participants with atopic dermatitis applied a lotion containing 1% of inactivated microbial extract to one side of their body and a placebo lotion to the other side for 28 days. No other creams were used simultaneously on the areas examined. The followed parameters were transepidermal water loss and erythema measurements, SCORAD index (scoring of AD), adverse effects, cytokines from skin tape strip samples and the skin microbiome. Microbial extract in lotion was well tolerated. It did not significantly affect atopic eczema severity, transepidermal water loss, erythema, total bacterial community composition or pro-inflammatory cytokines of atopic skin compared to placebo. Levels of the cytokines IL-1β, IL-22, IL-33, and the antimicrobial peptide hBD-2 decreased on atopic lesions, but this effect could not be separated from placebo. Daily use of diverse, inactivated microbial extract in lotion was well tolerated by participants with atopic dermatitis in a 28-day trial under supervision of a dermatologist. This method offers a novel and safe approach to introducing nature-based microbial exposure for healthy individuals and those with atopic dermatitis.