Carbon-based nanomaterials (CBN) are currently used in many biomedical applications. The research includes optimization of single grain size and conglomerates of pure detonated nanodiamond (DND), modified nanodiamond particles and graphene oxide (GO) in order to compare their bactericidal activity against food pathogens. Measurement of grain size and zeta potential was performed using the Dynamic Light Scattering (DLS) method. Surface morphology was evaluated using a Scanning Electron Microscope (SEM) and confocal microscope. X-ray diffraction (XRD) was performed in order to confirm the crystallographic structure of detonation nanodiamond particles. Bacteriostatic tests were performed by evaluating the inhibition zone of pathogens in the presence of carbon based nanomaterials. Raman spectroscopy showed differences between the content of the diamond and graphite phases in diamond nanoparticles. Fluorescence microscopy and adenosine-5′-triphosphate (ATP) determination methods were used to assess the bactericidal of bioactive polymers obtained by modification of food wrapping film using various carbon-based nanomaterials. The results indicate differences in the sizes of individual grains and conglomerates of carbon nanomaterials within the same carbon allotropes depending on surface modification. The bactericidal properties depend on the allotropic form of carbon and the type of surface modification. Depending on the grain size of carbon-based materials, surface modification, the content of the diamond and graphite phases, surface of carbon-based nanomaterials film formation shows more or less intense bactericidal properties and differentiated adhesion of bacterial biofilms to food films modified with carbon nanostructures.