Minimal inhibitory concentration (MIC) is the lowest antibiotic concentration that inhibits the visible growth of bacteria. Sub-minimal inhibitory concentration (Sub-MIC) is defined as the concentration of an antimicrobial agent that does not have an effect on bacterial growth but can alter bacterial biochemistry, thus reducing bacterial virulence. Many studies have confirmed that sub-MICs of antibiotics can inhibit bacterial virulence factors. However, most studies were focused on Gram-negative bacteria, while few studies on the effect of sub-MICs of antibiotics on Gram-positive bacteria. In this study, we examined the influence of sub-MICs of doxycycline, tetracycline, penicillin and amoxicillin on biofilm formation and coaggregation of Streptococcus gordonii, Streptococcus mutans, Actinomyces naeslundii, and Actinomyces odontolyticus. In this study, incubation with sub-MIC of antibiotics had no effect on the biofilm formation of S. gordonii and A. naeslundii. However, S. mutans showed increased biofilm formation after incubation with sub-MIC amoxicillin and penicillin. Also, the biofilm formation of A. odontolyticus was increased after incubating with sub-MIC penicillin. Coaggregation of A. naeslundii with S. gordonii and A. odontolyticus was diminished by sub-MIC amoxicillin. These observations indicated that sub-MICs of antibiotics could affect variable virulence properties such as biofilm formation and coaggregation in Gram-positive oral bacteria.