Objective Concentrated growth factor (CGF) is particularly gaining acceptance and popularity in regenerative dentistry. Nonetheless, there are no available studies showing its effect against microorganisms of oral cavity particularly in chronic oral disease-induced biofilms. This in vitro research was conducted to determine the antimicrobial effects of CGF against Staphylococcus aureus sp. (S. aureus) and Streptococcus mutans sp. (S. mutans). Materials and Methods Blood samples were obtained from a healthy volunteer. CGF was then prepared using specialized centrifugation equipment (Medifuge, Silfradent, Santa Sofia FC, Italy) and protocol. Antimicrobial activity of the CGF was observed and recorded on standard strains of S. aureus and S. mutans using a well diffusion method to determine the inhibition zone, broth microdilution to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), and crystal violet assay for biofilm assessment, with chlorhexidine (CHX) 0.12% used as a positive control. Statistical analysis was then performed using one-way analysis of variance followed by Tukey Test post hoc analysis. Results It was observed that there was a presence of clear zones of inhibition around the CGF after 24 hours of incubation. The mean diameter of the inhibition zone was 1.26 ± 0.12 nm and 1.20 ± 0.06 nm for S. aureus and S. mutans, respectively, with significance difference (p < 0.05) against the control group CHX 0.12%. The MIC values of the CGF against S. aureus and S. mutans were 47.9% and 34.17%, respectively, and the MBC values of the CGF against S. aureus and S. mutans were 100%. The viability and the ability in inhibiting the biofilm formation of S. mutans and S. aureus following treatment with CGF showed a reduction in the concentration-dependent manner as compared with the control group. Conclusion CGF possesses antimicrobial and antibiofilm activity against S. aureus and S. mutans.