The study investigated the antimicrobial activity of the essential oil extract of Ocimum gratissimum L. (EOOG) against multiresistant microorganisms in planktonic and biofilm form. Hydrodistillation was used to obtain the EOOG, and the analysis of chemical composition was done by gas chromatography coupled with mass spectrometry (GC/MS) and flame ionization detection (GC/FID). EOOG biological activity was verified against isolates of Staphylococcus aureus and Escherichia coli, using four strains for each species. The antibacterial action of EOOG was determined by disk diffusion, microdilution (MIC/MBC), growth curve under sub-MIC exposure, and the combinatorial activity with ciprofloxacin (CIP) and oxacillin (OXA) were determined by checkerboard assay. The EOOG antibiofilm action was performed against the established biofilm and analyzed by crystal violet, colony-forming unit count, and SEM analyses. EOOG yielded 1.66% w/w, with eugenol as the major component (74.83%). The MIC was 1000 µg/mL for the most tested strains. The growth curve showed a lag phase delay for both species, mainly S. aureus, and reduced the growth level of E. coli by half. The combination of EOOG with OXA and CIP led to an additive action for S. aureus. A significant reduction in biofilm biomass and cell viability was verified for S. aureus and E. coli. In conclusion, EOOG has relevant potential as a natural alternative to treat infections caused by multiresistant strains.