Controlling microbial growth is crucial for many biomedical, pharmaceutical and food industry applications. In this paper, we used a femtosecond laser to microstructure the surface of chitosan, a biocompatible polymer that has been explored for applications ranging from antimicrobial action to drug delivery. The influence of energy density on the features produced on chitosan was investigated by optical and atomic force microscopies. An increase in the hydrophilic character of the chitosan surface was attained upon laser micromachining. Patterned chitosan films were used to observe Staphylococcus aureus (ATCC 25923) biofilm formation, revealing an increase in the biofilm formation in the structured regions. Our results indicate that fs-laser micromachining is an attractive option to pattern biocompatible surfaces, and to investigate basic aspects of the relationship between surface topography and bacterial adhesion.