Introduction: Streptococcus suis is a zoonotic pathogen of great relevance to the swine industry, characterized by high genetic diversity and multiple serovars (SVs) with varying clinical prevalence. Biofilm formation represents a key factor in its virulence, antimicrobial resistance and infection persistence. Methods: We integrated gene expression profiling of biofilm-associated genes by RT-qPCR and antimicrobial susceptibility in planktonic and mature biofilm against five antibiotics in S. suis field isolates belonging to SV1, SV2, SV7 and SV9. Results: Expression of quorum sensing and adhesion genes (luxS, fbps, sadP and srtA) was significantly higher in SV2, the poorest biofilm formers, and inversely correlated with biofilm biomass, suggesting these factors act during early biofilm establishment. Correlation analysis indicated coordinated regulation among genes involved in quorum sensing, adhesion and capsule synthesis. Antimicrobial susceptibility testing revealed a high frequency of non-wild type phenotypes in planktonic cells for tetracycline, erythromycin and clindamycin (>80%), while ampicillin and ciprofloxacin were less frequent. Mature biofilms exhibited a significant increase in antimicrobial tolerance for all antibiotics tested, with SV2 showing the greatest susceptibility. Conclusions: These data highlight serovar-specific biofilm regulation patterns and enhanced drug tolerance in established S. suis biofilms.