Microalgal biofilm can remove nutrients and refractory pollutants from livestock wastewater, transforming them into biomass or fuel. However, these applications are hampered by biofilm shedding. Coculture enhances effects, and its influence on the adhesion of attached microalgal biofilms requires exploration. In this study, two unicellular algae, Euglena (E) and Chlorella pyrenoidosa (C). and two filamentous algae, Klebsodium sp. (K) and Phormidium sp. (P), were paired two by two, aiming to investigate the effect of mixing on the adhesion of microalgal biofilm. The results showed that suitable microalgal combinations significantly enhanced the performance of the co-cultured microalgal biofilm. Biomass enhancement of 35 % and 21.9 % was observed in the PC group compared to the individual P and C groups, respectively. The content of tightly bound extracellular polymeric substances (TB-EPS) and its protein and polysaccharide content significantly correlating with the net biomass increase in all treatments, p < 0.05. Furthermore, measurement of zeta potentials and water contact angles indicated that alterations in protein secondary structures contributed to a lower intermolecular repulsion and greater hydrophobicity of the TB-EPS, which together with the stability provided by the higher β-glucan content supported the adhesion of the co-cultured microalgal biofilm.