Group A streptococcus (GAS) is the primary cause of bacterial pharyngitis in children and adults. Up to one-third of patients treated for GAS pharyngitis fail to respond to antibiotic therapy. The objective of this cohort study was to evaluate GAS biofilm formation as a mechanism for antibiotic treatment failure using previously collected GAS isolates and penicillin treatment outcome data. The minimum biofilm eradication concentration (MBEC) assay device was used to determine the biofilm-forming capabilities, efficiencies, and antibiotic susceptibilities of GAS isolates. The MBECs and MICs of several antibiotics for GAS were determined. All 99 GAS isolates available for this study formed biofilms, with various efficiencies. Antibiotic MBECs were consistently higher than MICs for all of the GAS isolates. MBECs indicated penicillin insensitivity in 60% of GAS isolates, producing the first report of in vitro GAS insensitivity to penicillin. Using MBECs to predict penicillin treatment failure had better sensitivity (56%) but lower specificity (36%) than the sensitivity (0%) and specificity (100%) when MICs were used. However, the positive predictive value of the MBEC was superior to that of the MIC (56 versus 0%), while the negative predictive values (42 and 47%) were similar. More studies are needed to understand the roles of biofilms and the MBEC assay in predicting GAS treatment failure. In addition, further investigations are necessary to determine if non-biofilm-forming strains of GAS exist and the roles of in vivo monospecies and multispecies biofilms in streptococcal pharyngitis treatment failure.