Pretreatment of wool fabrics with chitosan and chitosan nanoparticles (1%, 2%, 3%) with synthesized direct dye is used to enhance the exhaustion of dye, fastness properties, and antimicrobial activity of dyed fabrics. Also, we use the ultrasonic technique for wool dyeing to reduce the temperature and time of dyeing. The results assessed for dyeing and antimicrobial activity indicate high-quality dyeing properties, However, direct dyes showed higher exhaustion and fixation values, fastness properties, and the colorimetric CIE L*a*b* C*h° data of the dyed wool fabrics. Also, these direct dyes have higher antibacterial activity toward Gram-positive and Gram-negative bacteria therefore they can be used in biomedical applications.