Background Functional fermented beverages are popular worldwide due to their potential to promote health. Starter culture is the main determinant of the final quality and flavor of fermented beverages. The co-cultivation of lactic acid bacteria (LAB) and yeast makes a significant contribution to the safe flavor of fermented beverages. However, the research on the potential of antioxidant, antimicrobial, and anti-biofilm formation of strawberry fermented beverage obtained by combining the LAB and yeast as starter cultures has not been well explored. Methods In this study, LAB and yeast were combined as starter culture to obtain strawberry fermented beverage. Fourier transform infrared (FTIR ) spectroscopy was used for the qualitative analysis of the fresh strawberry juice and fermented beverage. From the changes in antioxidant content, free radical scavenging ability, total superoxide dismutase (T-SOD) activity and total antioxidant capacity (T-AOC) to evaluate the antioxidant capacity of fermented beverage in vitro. The antibacterial ability was tested by the Oxford cup method. The biofilms of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538 under fermented beverages treatment was observed by Fluorescence microscope. In addition, sensory analysis was conducted in this study. Results In this study, the absorption peaks of Fourier transform infrared between 1,542cm.sup.-1 and 976 cm.sup.-1 , suggest the existence of organic acids, sugars and ethanol. The total phenols and total flavonoids content decreased by 91.1% and 97.5%, respectively. T-SOD activity increased by 33.33%.The scavenging ability of fermented beverage on superoxide anion free radicals was enhanced, and the scavenging ability on DPPH free radicals, hydroxyl free radicals, and ABTS free radicals was weakened. However, the T-AOC increased from 4.15±0.81 to 8.43±0.27 U/mL. Fermented beverage shows antibacterial activity against four pathogens. The minimum inhibitory concentration (MIC) values of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were 0.05 mL/mL and 0.025 mL/mL, respectively, and the minimum bactericidal concentration (MBC) were both 0.2 mL/mL. It was observed by fluorescence microscope that the green fluorescence area of the two biofilms is greatly reduced after being treated with fermented beverage. Sensory analysis results show that the average scores of fermented beverage in color, appearance and taste were increased. The overall impression and flavor were decreased. Conclusion These results demonstrated that strawberry fermented beverage has potential benefits such as an antioxidant, antibacterial, and anti-biofilm formation, providing the potential for the fermented beverage to become promising candidates for natural antioxidants, antibacterial agents and anti-biofilm agents.