Bacterial biofilms are involved in various medical infections and for this reason it is of great importance to better understand the process of biofilm formation in order to eradicate or mitigate it. It is a very complex process and a large range of variables have been suggested to influence biofilm formation. However, their internal importance is still not well understood. In the present study, a range of surface properties of Pseudomonas aeruginosa lipopolysaccharide mutants were studied in relation to biofilm formation measured in different kinds of multi-well plates and growth conditions in order to better understand the complexity of biofilm formation. Multivariate analysis was used to simultaneously evaluate the role of a range of physiochemical parameters under different conditions. Our results suggest the presence of serum inhibited biofilm formation due to changes in twitching motility. From the multivariate analysis it was observed that the most important parameters, positively correlated to biofilm formation on two types of plates, were high hydrophobicity, near neutral zeta potential and motility. Negative correlation was observed with cell aggregation, as well as formation of outer membrane vesicles and exopolysaccharides. This work shows that the complexity of biofilm formation can be better understood using a multivariate approach that can interpret and rank the importance of different factors being present simultaneously under several different environmental conditions, enabling a better understanding of this complex process.