In this study, we investigated the antibacterial effects of bilirubin on certain Gram-negative bacteria using both agar dilution and liquid microdilution methods. Twenty-five strains each of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa strains, isolated from various clinical samples in the medical microbiology laboratory of our hospital, were evaluated. Stock solutions of bilirubin (5 mg, 10 mg, and 15 mg/dl) (AppliChem, GmbH, Darmstadt, Germany) were prepared for agar dilution method. Aliquots of bilirubin stock solutions were added to liquid brain heart infusion medium in sterile microplates for liquid dilution methods. The average bilirubin MICs were 61.44, 62.72, ≥ 64.00, and ≥ 64.00 μg/mL for E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. In agar and liquid dilution methods, all tested bacteria grew at all bilirubin concentrations used. Bilirubin had no in vitro antibacterial effect on E. coli, K. pneumoniae, A. baumannii, or P. aeruginosa.