Diamond-like carbon films have unique properties for biological and medical applications due to their excellent biocompatibility, chemical inertness, and superior mechanical properties. In order to attend biomedical applications, there is an increasing interest in developing antibacterial coatings. In this paper, we investigated the bactericidal properties of diamond-like carbon films produced using plasma enhanced chemical vapor deposition. The films were deposited over 316L stainless steel substrates using a pulsed directly current discharge of methane gas. Diamond-like carbon structural quality was evaluated using Raman scattering spectroscopy. The bacterial adhesion and bactericidal activity of the coating was evaluated against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028 and Staphylococcus aureus ATCC 25923. These tests show diamond-like carbon bactericidal activity ranged between 25 and 55%, depending on the kind of bacteria. The bacterial adhesion on diamond-like carbon surface was influenced by its structure, chemical bonds and hydrogen content. The low surface roughness did not have decisive effect on its antibacterial performance.