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The development of self-cleaning biomimetic surfaces has the potential to be of great benefit to human health, in addition to reducing the economic burden on industries worldwide. Consequently, this study developed a biomimetic wax surface using a moulding technique which emulated the topography of the self-cleaning Gladiolus hybridus (Gladioli) leaf. A comparison of topographies was performed for unmodified wax surfaces (control), biomimetic wax surfaces, and Gladioli leaves using optical profilometry and scanning electron microscopy. The results demonstrated that the biomimetic wax surface and Gladioli leaf had extremely similar surface roughness parameters, but the water contact angle of the Gladioli leaf was significantly higher than the replicated biomimetic surface. The self-cleaning properties of the biomimetic and control surfaces were compared by measuring their propensity to repel Escherichia coli and Listeria monocytogenes attachment, adhesion, and retention in mono- and co-culture conditions. When the bacterial assays were carried out in monoculture, the biomimetic surfaces retained fewer bacteria than the control surfaces. However, when using co-cultures of the bacterial species, only following the retention assays were the bacterial numbers reduced on the biomimetic surfaces. The results demonstrate that such surfaces may be effective in reducing biofouling if used in the appropriate medical, marine, and industrial scenarios. This study provides valuable insight into the anti-fouling physical and chemical control mechanisms found in plants, which are particularly appealing for engineering purposes. [ABSTRACT FROM AUTHOR]