Abstract Interactions occurring between Saccharomyces cerevisiae and two thermostable α-amylase producing strains (Bacillus amyloliquefaciens 04BBA15 and Lactobacillus fermentum 04BBA19) were analyzed by comparing their growth patterns obtained in isolation with those obtained in mixture. The difference between the patterns was assessed using analysis of variance (ANOVA) in order to measure how much the growth of an organism was affected by other. The results showed two types of interactions in mixed culture; commensalism between S. cerevisiae and B. amyloliquefaciens 04BBA15 and mutualism between S. cerevisiae and L. fermentum 04BBA19. In mixed culture, the α-amylase production increased significantly compared to that observed in monoculture (P<0.05). Response surface optimization of fermentation parameters in mixed cultures (initial yeast to bacteria ratio 1.125, temperature 33.5°C, pH 5.5) resulted in about 1.8 fold higher enzyme production than that observed in the unoptimized fermentation.