The　microorganisms　in　biofilms　live　in　a　self-produced　matrix　of　hydrated　extracellular　polymeric　substances　(EPS)　that　forms　their　immediate　environment.　The　objective　of　this　paper　was　to　investigate　the　relationships　between　culture　conditions,　EPS,　microalgal　biofilms　growth　and　lipid　accumulation.　Fresh　water　alga　Botryococcus　braunii　was　attached　culture　in　multi-layers　photobioreactors　with　different　culture　media　and　substrates.　The　results　indicated　that　the　production　of　EPS　was　affected　by　culture　period,　nutrient,　and　substrate.　Increasing　the　production　of　EPS　may　enhance　the　biofilm　growth.　However,　the　EPS　components,　namely　proteins　and　polysaccharide　had　a　more　profound　effect　on　biofilm　formation　compared　to　total　EPS,　with　protein　being　more　significant　than　polysaccharide.　Nitrogen-free　and　EPS　strategies　were　conducted　to　increase　the　lipid　content　of　B.　braunii　biofilm　from　11.6　to　42.3　%　and　51.3　%,　respectively.　Compared　to　suspended　culture,　the　lipid　quality　was　enhanced.　The　dominant　component　of　hexadecanoic　acid　(16:0)　was　enhanced　from　21.78　to　48.17　%　and　55.44　%,　respectively.