Generally, the microorganism involved in soybean fermentation for the production of tempeh is Rhizopus oligosporus. However, Saccharomyces cerevisiae, a type of β-glucan-producing yeast, is known to be present and grow in the fermentation process. This study was aimed at investigating yeast and fungal growth dynamics, β-glucan formation, and antibacterial activity against Escherichia coli during the fermentation after adding S. cerevisiae as an inoculum. The Randomized Complete Block Design (RCBD) was applied with two treatments and three repetitions. Three types of starter culture were S. cerevisiae, R. oligosporus, and the combination of both. The second treatment was fermentation time at room temperature (
30
±
2
°
C
) for 0, 8, 16, 24, 32, and 40 hours. The dynamics were observed every eight hours. The obtained data were tested using Tukey’s Honestly Significant Difference (HSD) test. The results indicated that yeast grew during this process from a single S. cerevisiae culture and a mixture of R. oligosporus and S. cerevisiae, but not from R. oligosporus alone. The yeast grew during and until the end of fermentation and decreased after 32 hours in the mixed cultures. The β-glucan formed in tempeh with all types of inoculum, but the antimicrobial activity against E. coli increased with fermentation time and was significantly different between treatments. The highest β-glucan content and antibacterial activity of tempeh are from the mixed culture. In conclusion, the addition of S. cerevisiae and R. oligosporus in soybean fermentation produced tempeh with the highest β-glucan content and antibacterial activity against E. coli. The presence of β-glucans suggests higher health benefits of tempeh.