Veterinary diseases have significant economic repercussions, impacting livestock production, trade, and public health. These consequences include both direct expenses, such as veterinary treatments, vaccination initiatives, and animal losses, and indirect costs linked to reduced productivity and trade constraints. The management and prevention of these diseases require a multifaceted approach, involving biosecurity measures, vaccination strategies, and ongoing surveillance efforts. Many of the underlying causes of these veterinary diseases involve the formation of pathogenic biofilms. Biofilms, which are dense aggregates of microorganisms attached to surfaces, exhibit remarkable variations in their spatial and temporal organization, leading to a complex and dynamic ecosystem. Veterinary pathogens such as Brucella abortus, Corynebacterium pseudotuberculosis, Mycobacterium tuberculosis, Salmonella typhimurium, and Staphylococcus aureus are known to form biofilms in ruminants. Advances in mass spectrometry (MS) technologies have revolutionized proteomics studies, enhancing sensitivity, resolution, and throughput. Gel-based and liquid chromatography-based proteomics enable the precise identification and quantification of proteins within complex biofilm samples, thereby improving accuracy and comprehensiveness. This chapter discusses the potential application of a proteomics approach in deciphering the molecular mechanisms underlying virulence, antimicrobial resistance, interactions with host, and heterogeneity of veterinary biofilms. It provides a foundation for developing more effective treatments against these persistent and challenging bacterial communities.