Poly(ether-ether-ketone) (PEEK) has also shown to be very attractive for incorporating therapeutic compounds thanks to a sulfonation process which modifies the material structure resulting in a sulfonated-PEEK (sPEEK). Concerning biomedical applications, the objective of this work was to evaluate the influence of different sulfonation degree of sPEEK on the biofilm growth. PEEK samples were functionalized by using sulphuric acid (98%) and then dissolved into dimethyl-sulfoxide. A dip coating technique was used to synthesize sPEEK thin films. The sulfonation degree of the materials was analyzed by FT-IR, H NMR, TG and IEC. The surfaces were characterized by scanning electron microscopy, profilometry and contact angle analyses. Subsequently, the biofilm formation on sulfonated-PEEK based on Streptococcus mutans and Enterococcus faecalis was measured by spectrophotometry, colony forming units (CFUmL-1) and SEM. Results obtained from thermal and chemical analyses showed an intensification in sulfonation degree for sPEEK at 2 and 2.5h. The E. faecalis or S. mutans biofilm growth revealed statistically significant differences (p<0.05) between 2 and 3h sulfonation groups. A significant decrease (p<0.05) in CFUmL-1 was recorded for S. mutans or E. faecalis biofilm grown on 2.5 or 3h sPEEK. Regarding the thermal-chemical and microbiologic analyses, the sulfonation degree of sPEEK ranging from 2 up to 3h was successful capable to decrease the biofilm growth. That revealed an alternative strategy to embed anti-biofilm and therapeutic compounds into PEEK avoiding infections in biomedical applications.