Musalais is a very traditional Uygur ethnic characteristic drink and the co-existence of Saccharomyces cerevisiae and lactic acid bacteria (LAB) is widely presented in the natural fermentation process. Five LAB mediums were used for isolating from two Musalais samples and semi-quantitative assay was performed to examine the biofilm formation of the LAB strains. The positive-biofilm LAB was performed to mixed-cultivate with five Saccharomyces cerevisiae from Musalais. The effects of co-cultured with Saccharomyces cerevisiae on LAB growth and biofilm formation were explored and the effects of five Saccharomyces cerevisiae by the mixed biofilm formation on the ethanol fermentation were detected under the simulation conditions of Musalais natural fermentation. The results showed that 21 suspected LAB strains were isolated from two Musalais samples and two strains were detected as strongly positive biofilm formation (OD490nm>0.56). The further identification results showed that the two strains were Lactobacillus plantarum (T1-8) and Pediococcus pentosaceus (M1-6), respectively. After co-culturing, the Saccharomyces cerevisiae A had a very significant effect on the biofilm formation of the L.plantarum (T1-8) （p＜0.01) and the Saccharomyces cerevisiae B and D had significant effects on the biofilm formation of the L.plantarum (T1-8) （p＜0.05). However, all the Saccharomyces cerevisiae had no obvious effects on growth and biofilm formation of P.pentosaceus (M1-6). The ethanol yields of the corresponding Saccharomyces cerevisiae were improved nearly twice as much or more in the groups of A+T1-8, B+T1-8, C+T1-8, D+T1-8, B+M1-6, C+M1-6 and D+M1-6. The other groups also had different degrees of increase.