Benthic diatoms may secrete large amounts of extracellular polymeric substances which constitute the basic compounds of photoautotrophic biofilms. Such natural biofilms harbor microbial communities in which, besides diatoms, bacteria are considered to be important members that remineralize organic matter, thereby supporting algal growth. In order to study biofilm formation of diatoms in the presence of bacteria, we established and optimized a co-culture system comprising the model diatom Phaeodactylum tricornutum and the marine bacterium Roseovarius sp. strain 217. We found that an initial pH of 8 increases biofilm formation of P. tricornutum within the first 3 days, while repressing biofilm formation of axenic R217. Omission of medium buffer substances allows a pH elevation during cultivation, further enhancing biofilm formation by P. tricornutum. Furthermore, we found that an initial bacterium:diatom ratio of 100:1 was found to be optimal for biofilm formation in this co-culture system. A major finding is that the bacteria-free spent medium of R217 also can increase P. tricornutum biofilm formation, indicating that soluble bacterial compounds may act as signaling molecules, which allows using this biofilm system for experiments requiring bacteria-free conditions.