Recycling spent lithium-ion batteries is important from an environmental perspective and metals worthiness. In this study, an ultrasonic-assisted bioleaching process was developed to recycle valuable metals from spent lithium- ion batteries using bacterial supernatant of Acidithiobacillus ferrooxidans. The results showed that 13% Co, 42% Mn, 25% Ni, and 57% Li were extracted after 24 hours from 10 g/L pulp density without ultrasonication. Ultrasonication with a power of 203.5W for 30 minutes with intervals of 1 hour enhanced the metal extraction and reduced bioleaching time; 19% Co, 50% Mn, 34% Ni, and 67% Li were extracted after 12 hours from 10 g/L pulp density. This study also investigated the effect of ultrasound on the growth of the pure culture of Acidithiobacillus ferrooxidans. Results indicated that a 10-minute daily application of ultrasonic waves with a power of 203.5W was optimal for bacterial growth. The results of metals extraction and morphological, structural, and elemental analyses of the battery powder before and after the bioleaching process proved that the application of ultrasound effectively enhanced metal extraction and reduction of bioleaching time as well as the growth of Acidithiobacillus ferrooxidans.