Irradiation with deep-ultraviolet light-emitting diodes (DUV LEDs) is emerging as a low energy, chemical-free approach to mitigate microbial contamination, but the effect of surface conditions on treatment effectiveness is not well understood. Here, inactivation of L. innocua and E. coli ATCC25922, as examples of Gram-positive and Gram-negative bacteria, respectively, by DUV LED of 280 nm wavelength was studied. Surface scenarios commonly encountered in environmental, clinical or food processing environments were used: nutrient rich surfaces, thin liquid films (TLF), and stainless steel surfaces (SS). DUV LED exposure achieved 5-log reduction for both strains within 10 min in most scenarios, except for TLF thicker than 0.6 mm. Inactivation kinetics in TLF and on dry SS followed the Weibull model (0.96 ≤ R 2 ≤ 0.99), but the model overestimated inactivation by small-dose DUV on wet SS. Confocal microscopy revealed in situ that bacteria formed a dense outer layer at the liquid-air interface of the liquid droplet, protecting the cells inside the droplet from the bactericidal DUV. This resulted in lower than anticipated inactivation on wet SS at small DUV doses, and deviation from the Weibull model. These findings can be used to design effective DUV LED disinfection strategies for various surface conditions and applications. Persistence of pathogens on material surfaces often causes severe consequences, including infections in dental offices and hospitals 1 , or transfer of pathogenic or spoilage microorganisms from food contact surfaces to food products in food processing facilities and food service environments 2. Exposing surfaces contaminated by microorganisms to ultraviolet (UV) of wavelength 100-280 nm has been established as an effective disinfection method, often used as an alternative to or in tandem with chemical disinfection methods. Mercury lamps are currently the most commonly used source of UV light. Yet, according to the Minamata Convention on Mercury 3 , signed in 2013, manufacturing and trading of mercury-containing lamps for general lighting purposes will be disallowed after 2020, to reduce and eliminate the adverse effects of mercury on human health and the environment. This agreement accelerated the efforts for the development of alternatives to mercury lamps. Light-emitting diodes that emit light in the UV range (UV LEDs) present several advantages compared to mercury lamps, including the lack of toxic mercury, device compactness and flexible designs, zero warm-up time 4,5 , high durability, monochromatic light emission at specific wavelength 6 , wavelength diversity, possibility of pulsed illumination, and the capability of maintaining relatively high activity at cold temperatures (e.g. refrigeration) 5. UV LEDs are also known for their low heat emission in the form of IR radiation 7 , which enables applications that demand high UV fluence while preventing heating over long periods of time. Recent progress in improving the light-extraction efficiency of UV LEDs in the range 200-300 nm has increa...