This study examined the microbicidal activity of ultraviolet (UV)-C185-256-nm irradiance (robot 1) and ozone generated at UV-C185-nm by low-pressure mercury vapor lamps (robot 2) adapted to mobile robotic devices for surface decontamination, which was achieved in less than 1 h. Depending on their wall structure and outer envelopes, many microorganisms display different levels of resistance to decontaminating agents. Thus, the need for novel disinfection approaches is further exacerbated by the increased prevalence of multidrug-resistant bacteria, as well as the potential of novel microorganisms, with the ability to cause disease outbreaks. To set up a rapid and effective approach for microorganisms propagation prevention, we focused on the effects of UV-C and ozone on a distinct microorganism survival ratio. A set of microorganisms, including Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, Trichoderma harzianum, and Bacillus subtilis, were used to evaluate the disinfection power of UV-C and UV-C plus ozone generating robots. UV-C disinfection can be suited to ad hoc tasks, is easy to operate, requires low maintenance, does not have the need for the storage of dangerous chemicals, and does not produce by-products that may affect human health and the environment. The robotic cumulative irradiation technology developed (fluence accumulated values of 2.28 and 3.62 mJ cm-2, for robot 1 and 2, respectively), together with the production of ozone (with a maximum peak of 0.43 ppm) capable of reaching UV-C shaded surfaces, and analyzed in the current study, despite being designed for the need to reduce the risk of epidemic outbreaks in real-life scenarios, represents a versatile tool that could be employed for air and surface disinfection within many circumstances that are faced daily.