Tempe is a traditional Indonesian fermented soybean mostly produced in small industries and sold locally throughout the country. Studies on the bioactive peptides in tempe are rare. Here, we studied bioactive peptides in samples from three tempe producers with different degrees of sanitation. The peptide sub-fractions of tempe from each producer were collected following water extraction, ultrafiltration (<3 kDa), gel filtration chromatography, and reversed phase high performance liquid chromatography (RP-HPLC) separation followed by liquid chromatography-mass spectrometry (LC-MS). The MS spectra were then predicted using FindPept tools, and their biofunctionalities were confirmed with BIOPEP databases. There were few similar peptides found in tempe from the three producers. Peptides Val-His and Ala-Leu-Glu-Pro were found in tempe from all producers. Producers having a good sanitation level had more bioactive peptides than those with moderate or poor sanitation levels (58%, 43% and 35%, from good to poor sanitation). This work showed that the tempe from the three producers had antihypertensive, antidiabetic, antioxidative and antitumor peptides. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.