Allyl isothiocyanate (AITC) is a natural compound known for its potential antimicrobial properties. In this study, we present a comprehensive investigation into the isolation, characterization, and antimicrobial activity of AITC which is obtained from mustard seeds. The isolation of AITC was achieved through an efficient extraction process, employing extraction assembly. Subsequently, AITC was subjected to a series of physicochemical tests to elucidate the presence of physicochemical constituents. Furthermore, we employed UV-visible spectroscopy and infrared (IR) spectroscopy to obtain valuable insights into the structural features of AITC. To gain a deeper understanding of the phyto-constituents present in AITC, gas chromatography-mass spectroscopy (GC-MS) was used for phytochemical profiling. The antimicrobial activity of AITC was assessed against two pathogenic microorganisms, Staphylococcus and Escherichia coli. The outcomes of our study reveal the possible potential of AITC as an antimicrobial agent, demonstrating its inhibitory effects on the growth of both microbial strains and zone diameter of inhibitions are 12, 10, and 4 mm for Staphylococcus and 14, 12 and 6 mm for E. coli and hence the AITC extracts were found to have antibacterial activity. This research provides treasured comprehensive insights into the isolation, characterization, and antimicrobial belonging properties of AITC which is derived from mustard seeds. The findings suggest that AITC has promising applications in the field of antimicrobial research, opening avenues for further investigation into its potential therapeutic uses.