Low density polyethylene (LDPE) based nanocomposites containing multi-walled carbon nanotubes (MWCNTs) were prepared by a two-step process consisting of a pre-mixture using high energy ball milling (HEBM) and subsequent hot pressing. The effects of ball milling and the presence of the MWCNTs on some physical properties of the materials and the antimicrobial efficiency against DH5 Escherichia coli were studied. Fourier transform infrared spectroscopy revealed that the polymer structure did not change in the final materials after the addition of MWCNTs and mixing. Differential scanning calorimetry showed small differences in the LDPE thermal behavior as a function of the type of material due to small changes in the polymer crystallization. This result was mainly ascribed to the milling process rather than to the incorporation of the MWCNTs. The presence of 1% by weight of the nanofiller increased the rigidity and hydrophobicity of the nanocomposites with respect to neat LDPE. This effect was explained by the preferential location of the MWCNTs in the surface of the material as the main factor decreasing the polar contribution to the surface free energy. A correlation between hydrophobicity, biofilm development and the shape and size of DH5 E. coli was observed, indicating that the presence of MWCNTs leads to a biocide effect by decreasing cell adhesion and changing its metabolism.