Naphthalene-1,4-dione derivatives were synthesized and tested against selected bacterial strains. All the tested compounds were prepared by direct introduction of corresponding substituents into the naphthoquinone core in oxidative conditions. In this study, eight strains of bacteria (Proteus, Escherichia, Klebsiella, Staphylococcus, Enterobacter, Pseudomonas, Salmonella, Enterococcus) were used for determination of antimicrobial activity of synthesized compounds with the Minimal Inhibitory Concentration (MIC) method. Additionally, selected compounds were tested for haemolytic activity using human erythrocytes. All naphthalene-1,4-dione derivatives exhibited significant antimicrobial activity with MIC values between 7.8 and 500 μg/ml. A majority of the synthesized compounds showed the strongest antibacterial properties towards S. aureus, with a high level of selectivity. None of the tested naphthalene-1,4-dione derivatives exhibited haemolytic activity.