ABSTRACTAcinetobacter baumanniiis a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) ofA. baumanniican withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process inA. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease inA. baumanniiaffects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing keyA. baumanniiprocesses to survive stress and to maintain viability.IMPORTANCEAcinetobacter baumanniiis an opportunistic pathogen and is a leading cause of hospital-acquired infections.A. baumanniiis difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation inA. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon inA. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology ofA. baumannii, which will permit us to find effective ways to eliminate the bacterium.