The co-existence of Saccharomyces cerevisiae and Lactobacillus widely exists in fermented food and their co-existence endows fermented foods with quite different flavors and tastes. Meanwhile, a large number of active substances and prebiotic substances were added to the fermented foods and they were very beneficial to human health. Five Saccharomyces cerevisiae strains and six Lactobacillus strains from different fermented foods were used in the research. Semi-quantitative assay was performed to examine effects of Lactobacillus co-cultured with Saccharomyces cerevisiae and the metabolites on Lactobacillus growth and biofilm formation. The results showed that all of the five Saccharomyces cerevisiae strains could promote Lactobacillus growth but had different effects on biofilm formation. All of the Saccharomyces cerevisiae metabolites did not have obvious effects on Lactobacillus growth but also had effects on biofilm formation in different degrees. Especially, extremely significant promotion was found in the co-existence groups of E+1, B+3 and E+3, and extremely significant inhibition was also found in the co-existence groups of B+1, D+9 and E+9. It may be due to the strict selection roles of each other between the two strains. Meanwhile, comparing co-existence groups with metabolites groups, there was no very obvious connection. It appeared that different active substances acted different roles in the co-existence groups and metabolites groups.