Biomechanics and mechanobiology have long been recognized as essential for the growth and function of biological systems, and recent work has demonstrated the importance of mechanical forces for key physiological mechanisms in bacteria. Although the bacterial cell envelope is the primary load-bearing structure of bacteria, the influence of mechanical stress on the bacterial cell envelope and its components is largely understudied. In this thesis I examine the role of mechanical stress on two systems in the bacterial cell envelope: multicomponent efflux complex MacAB-TolC which contributes to antibiotic resistance in Escherichia coli and two-component signaling system VxrAB which controls gene expression for cell wall synthesis in Vibrio cholerae.Multicomponent efflux complexes form a channel through the bacterial cell envelope in order to pump toxins and antibiotics out of the cell. We have previously shown that mechanical stress compromises the assembly and functionality of efflux complex CusCBA; however, it is unknown if other efflux complexes are similarly vulnerable to mechanical stress and the role cell envelope stiffness plays. We expand upon previous work by investigating the influence of mechanical stress on efflux complex MacAB-TolC with and without alterations to cell envelope stiffness. We submitted individual live bacterial cells to controlled mechanical loading using a custom microfluidic device and used single-molecule tracking to observe efflux pump behavior. We found that octahedral shear stress in the cell envelope promotes efflux complex disassembly, suggesting impaired antibiotic resistance capability. Cell envelope stiffness plays a significant role in mediating the effect of mechanical manipulation through the magnitude of octahedral shear stress as well as changes in cell surface area. Our findings demonstrate the importance of mechanical stress in the cell envelope as well as cell envelope stiffness for trans-envelope protein function. Although the bacterial cell envelope is the load-bearing component of the cell, it is unknown if cell envelope homeostasis is responsive to mechanical stress. VxrAB is a two component signaling system with a sensor embedded in the cell envelope and a response receptor that controls gene expression of cell wall synthesis. We submitted cells to mechanical loading using our microfluidic device, hydrostatic pressure, and compression and measured the activity of the VxrAB signaling system in response. We found that cells experiencing greater magnitudes of mechanical load exhibited greater VxrAB signaling. Our results suggest the importance of mechanical signals in cell envelope homeostasis through VxrAB mediated cell wall synthesis. Together, this work suggests the importance of mechanical stress for the function of proteins in the bacterial cell envelope. This work establishes a foundation for future bacterial mechanobiology research and has the potential to advance synthetic biology as well as inform future antibiotic treatment strategies.