Optical tweezers (OT) are a technique that, by focused laser light, can both manipulate micrometer sized objects and measure minute forces (in the pN range) in biological systems. The technique is therefore suitable for assessment of bacterial adhesion on an individual adhesin-receptor and single attachment organelle (pili) level. This chapter summarizes the use of OT for assessment of adhesion mechanisms of both non-piliated and piliated bacteria. The latter include the important helix-like pili expressed by uropathogenic Escherichia coli (UPEC), which have shown to have unique and intricate biomechanical properties. It is conjectured that the large flexibility of this type of pili allows for a redistribution of an external shear force among several pili, thereby extending the adhesion lifetime of bacteria. Systems with helix-like adhesion organelles may therefore act as dynamic biomechanical machineries, enhancing the ability of bacteria to withstand high shear forces originating from rinsing flows such as in the urinary tract. This implies that pili constitute an important virulence factor and a possible target for future anti-microbial drugs.