The pathogenesis of chronic hand eczema remains un-clear. Insights into the skin microbiome in hand eczema and its potential relevance to disease severity may help to elucidate the underlying mechanisms of hand eczema. The aim of this study was to characterize the microbiome in patients with hand eczema and healthy controls. A 5-visit prospective study was conducted over a period of 3 weeks. At each visit, bacterial swabs were taken from the hands of patients with hand eczema and controls. The microbiome was examined using DNA extraction and 16S rRNA amplicon sequencing (V3–V4 regions). Fifty patients with hand eczema and 50 controls were included (follow-up rate=100%). The baseline bacterial α-diversity was reduced on the hands of patients with hand eczema compared with controls (effect size=–0.31; 95% confidence interval (95% CI) –0.50; –0.11; p = 0.003). The dysbiosis on the patients’ hands was stable over the study period, was associated with disease severity, and was characterized by reduced bacterial diversity and different bacterial community compositions.