Microalgae biofilm-based culture systems have wide applications in environmental engineering and biotechnology. Biofilm structure is critical for the transport of nutrients, gas, and signaling molecules in a microalgal biofilm. This work aims to understand the influence of cell surface energy (SE) on the microalgal biofilm structure. Three microalgae species were used as model cells in the study: Chlorella sp., Nannochloris oculata, and Chlorella pyrenoidosa. First, by mediating biofilm culture conditions, we obtained Chlorella sp. cells with SEs of 40.4 +/- 1.5, 44.7 +/- 1.0, and 62. 7 +/- 1.2 mJ/m(2), N. oculata cells with SEs of 47.7 +/- 0.5, 41.1 +/- 1.0, and 62.6 +/- 1.2 mJ/m(2), and C. pyrenoidosa cells with SEs of 64.0 +/- 0.6, 62.1 +/- 0.7, and 62.8 +/- 0.6 mJ/m(2). Then, based on the characterizations of biofilm structures, we found that cell SE can significantly affect the microalgae biofilm structure. When the cell SEs ranged from 40 to 50 mJ/m(2), the microalgae cells formed heterogeneous biofilms with a large number of open voids, and the biofilm porosity was higher than 20%. Alternatively, when the cell SEs ranged from 50 to 65 mJ/m(2), the cells formed a flat, homogeneous biofilm with the porosity lower than 20%. Finally, the influencing mechanism of cell SE on biofilm structure was interpreted based on the thermodynamic theory via analyzing the co-adhesion energy between cells. The study has important implications in understanding factors that influence the biofilm structures.