Tempeh is traditionally produced by fermenting soybean with the fungus Rhizopus oligosporus found in banana leafs. We wanted to investigate if Taiwan's flavorful red bean could be used as a healthy substitute for soybeans in tempeh. One bioactive component of tempeh is γ-Aminobutyric acid (GABA). We measured GABA content and shelf-life-related antimicrobial activity in red-bean tempeh made with four strains of Rhizopus, one purchased strain of Rhizopus, and an experimental co-cultured group (Rhizopus and Lactobacillus rhamnosus BCRC16000) as well as cortisol in red-bean-tempeh-treated zebrafish. GABA was highest in the co-culture group (19.028 ± 1.831 g kg-1), followed by screened Strain 1, the purchased strain, and screened Strain 4. All strains had antibacterial activity on S. aureus and B. cereus. The extract significantly reduced cortisol in zebrafish. However, Strain 1, with less GABA than some of the other strains, had the best effect on cortisol level, suggesting that other components in red-bean tempeh may also affect stress-related cortisol. We found the benefits of red-bean tempeh to be similar to those reported for soybean-produced tempeh, suggesting that it could be produced as an alternative product. Considering the Taiwanese appreciation of the red-bean flavor, it might find a welcoming market.