This study was aimed to investigate the structure of bioactive components of black seed oil (BSO) and their antimicrobial and cytotoxic effects. Initially, the structural examination was conducted using various spectroscopic techniques, such as FTIR, TLC, and UV‐visible spectroscopy, which are important in determining substituents, functional groups, and the presence of conjugated double bonds in BSO. From the FTIR spectra, a variety of sharp, strong, and weak peaks were specified relating to the main components of thymoquinone (TQ), dithymoquinone, thymohydroquinone, and thymol in BSO. The results of UV‐visible spectroscopy confirmed the presence of thymoquinone as a major compound, and conjugated double bonds were also found. In addition, qualitative TLC analysis was used to identify thymoquinone from the methanol‐extracted layer in BSO, by calculating the retention factor (R_f) value. Furthermore, antimicrobial activity of BSO was studied against various types of bacteria. Strong bacterial inhibitory effects were observed, especially against Bacillus subtilis, with an average inhibition zone of 15.74 mm. Moreover, through the use of the MTT assay in vitro, it was shown that BSO does not exhibit any cytotoxicity towards human peripheral blood mononuclear cells (PBMCs). It was also found from the structural characterization of BSO that the existence of TQ is responsible for potential antibacterial activity without any cytotoxic effects. The main observation of this work is that BSO has antimicrobial activity even against methicillin‐resistant Staphylococcus aureus (MRSA).