This review describes the principles for generation of affibody molecules using bacterial display on the Gram-negative Escherichia coli and the Gram-positive Staphylococcus carnosus, respectively. Affibody molecules are small and robust alternative scaffold proteins that have been explored for therapeutic, diagnostic, and biotechnological applications. They typically exhibit high-stability, affinity, and specificity with high modularity of functional domains. Due to the small size of the scaffold, affibody molecules are rapidly excreted through renal filtration and can efficiently extravasate from blood and penetrate tissues. Preclinical and clinical studies have demonstrated that affibody molecules are promising and safe complements to antibodies for in vivo diagnostic imaging and therapy. Sorting of affibody libraries displayed on bacteria using fluorescence-activated cell sorting is an effective and straightforward methodology and has been used successfully to generate novel affibody molecules with high affinity for a diverse range of molecular targets.