Black cumin (Nigella sativa L.) oil has been traditionally used to manage infectious diseases. The scientific validation of its antibacterial potential remains of significant pharmacological interest. This study evaluated the in vitro antibacterial activity of cold-pressed black cumin oil against selected bacterial strains and compared its efficacy with that of common β-lactam antibiotics, supplemented by mechanistic insight through molecular docking. Pure oil was obtained via cold-press extraction from seeds. Antibacterial activity was evaluated using the disk diffusion and broth microdilution methods against Staphylococcus aureus NCTC10788, Bacillus cereus NCTC7464, Listeria monocytogenes ATCC11994, Escherichia coli NCTC2001, and Salmonella typhimurium NCTC11994. Commercial antibiotic disks containing cloxacillin (5 µg), cefoperazone (75 µg), penicillin (40 µg), and amoxicillin (25 µg) served as a reference. Potential molecular interactions were explored by the density functional theory (DFT) optimization of thymoquinone at the B3LYP/6-31+G(d,p) level, followed by molecular docking against bacterial targets. Inhibition zone diameters ranged from 13.5 ± 0.7 mm to 34 ± 2.1 mm, and minimum inhibitory concentration (MIC) values varied between 6.7 ± 2.3 and 64 ± 0.0 µg/mL depending on the bacterial strain tested. Black cumin oil demonstrated a stronger inhibitory effect on B. cereus and L. monocytogenes than the other bacteria tested, and exhibited a significantly higher inhibitory effect than some of the antibiotics tested (p < 0.05). In contrast, no statistically significant differences were observed among treatments against E. coli (p > 0.05). Overall, Gram-positive bacteria showed greater susceptibility to black cumin oil than Gram-negative bacteria. The computational analyses demonstrated stable binding interactions supporting the experimental results. These integrative in vitro and in silico findings provide mechanistic evidence for the traditional use of black cumin oil in treating infections. The results suggest that black cumin oil could be a promising natural antibacterial candidate; however, further toxicological and pharmacokinetic evaluations are required prior to clinical use.