The modern era has raised awareness of healthier lifestyles and food safety, creating new goals for scientists and industry. Here, pathogen microorganisms represent one of the difficulties in ensuring food quality. Microorganism’s resistance to broad-spectrum antibiotics has increased, especially with the planktonic cells' ability to switch into sessile mode and form biofilms. In the endeavor for efficient natural food preservatives, essential oils represent one of the possible solutions for controlling food spoilage, due to their recognized antimicrobial properties. In this study, the assessment of the chemical profile of Piper nigrum L. essential oil (PNEO) was performed. The GC-MS analysis identified (E)-caryophyllene, limonene, and sabinene as prevalent constituents of PNEO, which prompted further antimicrobial and antibiofilm investigations. The minimal inhibitory concentration (MIC) assays revealed that PNEO generally expressed better antimicrobial properties in comparison to the common antibiotics. Moreover, the in situ antimicrobial analysis uncovered the ability of the PNEO’s vapor phase to inhibit the growth of L. monocytogenes, S. aureus, and H. influenzae on selected food models, exposing PNEO as a promising preservation agent. Antibiofilm effects of PNEO were evaluated against biofilm-forming P. fluorescens and S. maltophilia employing MIC assays, crystal violet assays, MALDI-TOF MS analysis, and molecular docking. The obtained results in MIC and crystal violet assays indicated significant antibiofilm activity of PNEO. The MALDI-TOF MS analysis revealed that PNEO altered the protein profiles of the investigated bacteria on stainless steel and glass surfaces, especially in the case of S. maltophilia. In silico antibiofilm investigations indicated a potential involvement of the PNEO components in the interference of quorum-sensing and drug-resistance mechanisms. These findings underline the antimicrobial and antibiofilm virtues of PNEO, indicating its promising usage in food preservation.