Aims To investigate the biofilm inhibitory activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. Methods and Results The pulp of red pitahaya and the leaves of red spinach were extracted using methanol followed by subfractionation to obtain betacyanin fraction. The anti-biofilm activity was examined using broth microdilution assay on polystyrene surfaces and expressed as minimum biofilm inhibitory concentration (MBIC). The betacyanin fraction from red spinach showed better anti-biofilm activity (MBIC: 0 center dot 313-1 center dot 25 mg ml(-1)) against five Staph. aureus strains while the betacyanin fraction from red pitahaya showed better anti-biofilm activity (MBIC: 0 center dot 313-0 center dot 625 mg ml(-1)) against four P. aeruginosa strains. Both betacyanin fraction significantly reduced hydrophobicity of Staph. aureus and P. aeruginosa strains. Numbers of Staph. aureus and P. aeruginosa attached to polystyrene were also reduced without affecting their cell viability. Conclusion Betacyanins can act as anti-biofilm agents against the initial step of biofilm formation, particularly on a hydrophobic surface like polystyrene. Significance and Impact of the Study This study is the first to investigate the use of betacyanin as a biofilm inhibitory agent. Betacyanin could potentially be used to reduce the risk of biofilm-associated infections.