ABSTRACTDipeptides were constructed using hydrophobic amino acid residues following AMP prediction. After that Boc‐modification was performed on the screened peptides and finally Boc‐Phe‐Trp‐OMe and Boc‐Trp‐Trp‐OMe were synthesized. Even though no inhibition zones were observed in agar well diffusion assays, minimum inhibitory concentration (MIC) analysis revealed anti‐bacterial activity against both Gram‐positive and Gram‐negative bacteria, with MIC90 ranging from 230 to 400 μg/mL. The crystal violet assay confirmed the dipeptides' biofilm eradication and disruption capabilities. Furthermore, membrane permeabilization assays indicated outer and inner membrane permeabilization, while SEM analysis revealed the formation of fibril and spherical nanostructures, likely contributing to this effect. The peptides also exhibited resistance to protein adsorption, non‐cytotoxicity, and non‐hemolytic properties, making them promising broad‐spectrum anti‐bacterial agents with biofilm eradication and disruption potential. This study concludes that Boc‐protected phenylalanine‐ and tryptophan‐based dipeptides can self‐assemble and can be used as broad‐spectrum anti‐bacterial agents. The self‐assembly of these peptides offers a versatile platform for designing biomaterials with tailored properties and functionalities. Research exploring the anti‐bacterial potential of Boc‐protected dipeptides has been limited, prompting our investigation to shed light on this overlooked area. Our analysis of synthesized Boc‐protected dipeptides revealed notable anti‐bacterial activity, marking a significant advancement. This finding suggests that these dipeptides could emerge as potent, broad‐spectrum anti‐bacterial agents, addressing the urgent need for effective treatments against bacterial resistance and opening new avenues in therapy. This study not only enhances our understanding of these dipeptides but also highlights their potential as innovative and efficacious anti‐bacterial agents, making a substantial impact in the clinical field.