Broiler processing environments are a source of spoilage bacteria and potential pathogens. The aim of this study was to investigate the impact of cleaning and disinfection (C&D) on bacterial load and diversity in two broiler processing plants and to determine the efficacy of industry-standard disinfectants (DIs). C&D significantly reduced average bacterial loads on surfaces from 3.7 to 1.8 log CFU/cm in Plant A and from 7.0 to 3.8 log CFU/cm in Plant B ( < 0.001). Metataxonomics revealed that and an unknown Enterobacteriaceae genus dominated before C&D in Plants A and B, respectively, while was predominant after C&D in both plants. Bacterial diversity only declined significantly after C&D in Plant B. Bacterial loads also declined across hygienic zones along the broiler processing line in Plant A. During slaughter, , , and dominated, and bacterial loads were significantly higher compared with post-slaughter zones, which were dominated by . Culture-dependent analyses confirmed the presence of spp. and also bacteria affecting food safety, including , , , , and . A selection of these was tested against DIs using robotic high-throughput screening. At the recommended user concentrations, DIs effectively inhibited planktonic bacteria and significantly reduced mono-species biofilms. However, none of the DIs completely eradicated all biofilms at these concentrations, with survival rates ranging from 7% to 53%, depending on the DI. In conclusion, C&D effectively reduces the bacterial burden and reshapes the bacterial microbiota with incomplete biofilm eradication by commercial DIs.IMPORTANCEBroiler meat continues to be involved in bacterial disease outbreaks. The surface microbiota in broiler processing environments can be a source of contaminating bacteria. Our study highlights the importance of effective C&D routines since potential pathogens and spoilage bacteria are found in these environments. Furthermore, the study provides evidence of biofilms surviving high concentrations of industry-standard DIs. This emphasizes the importance of additional measures to facilitate biofilm removal, such as mechanical cleaning, but also suggests that there is a need for DIs with stronger biofilm eradication capabilities. Ultimately, it is important to understand and continuously improve the state of hygiene in broiler processing plants to mitigate the risk of foodborne disease outbreaks.