In this study, the focus was on the effects of sub-MICs of the antibiotics on adherence, hydrophobicity, and biofilm formation by two groups of Streptococcus pyogenes strains, which were responsible for different clinical cases. The aim of this study was to explore the effects of sub-MICs of penicillin, ceftriaxone, erythromycin, and clindamycin on adherence, surface hydrophobicity, and biofilm biomass in two selected collections of group A streptococcus (GAS): strains isolated from carriers (CA) and strains isolated from patients with tonsillopharyngitis (TPh). Isolates were tested for hydrophobicity to xylene, adherence, and biofilm production in uncoated microtiter plates before and after treatment with 1/2 and 1/4 MICs of antibiotics. Penicillin reduced adherence and biofilm production in TPh strains, whereas ceftriaxone diminished adherence and biofilm formation in CA group. On the contrary, clindamycin enhanced adherence and biofilm production in both groups of strains. Erythromycin did not significantly alter adherence, but triggered biofilm production in both groups of isolates. Hydrophobicity of both groups of strains was significantly reduced after exposure to all antibiotics. Beta-lactams displayed anti-biofilm activity; penicillin diminished both adherence and biofilm production in TPh strains, whereas ceftriaxone reduced it in strains isolated from CA.