Summary In the present study, the effects of continuous and pulsed ultrasound (100 W, 30 kHz, 25–75% amplitudes for 30 min) and fermentation process (37°C for 24 h) by Lactobacillus helveticus PTCC 1332 on biological activities of Chortan were investigated. The obtained results showed that fermentation enhanced protease content (36.27% (neutral), 37.57% (acid), 39.28% (alkaline) protease), peptide concentration (45.27%) and antioxidant activity (33.69% (DPPH), 132.85% (ABTS)) of samples compared to unfermented ones. Additionally, an increase in ultrasound amplitude (except for continuous at 75%) promoted the protease enzymes, peptides and radical scavenging activity of the fermented and unfermented samples. Our results indicated that the ultrasound and fermentation treatments complemented each other. Depending on the experimental conditions, the effect of continuous ultrasound on samples was sometimes lower, sometimes similar and sometimes higher than that of pulsed treatment. Therefore, selection of an appropriate ultrasound mode and amplitude can improve the biological activity of Chortan.