Abstract: This study investigated the impact of antimicrobial peptide polylysine (ε-PL) on collagen (Col) self-assembly under physiological conditions. The results indicated that the cationic ε-PL had a decelerating effect on the collagen self-assembly process, leading to a looser network structure and a decrease in the fibril diameter of collagen assemblies. When the Col/ε-PL ratio was below 1∶0.5, well-defined collagen fibrils with D-periodicity were observed, and the thermal stability of the Col/ε-PL assemblies was similar to that of pure collagen assemblies. Moreover, the introduction of ε-PL also enhanced the antimicrobial activity of the Col/ε-PL assemblies. Therefore, incorporating an optimal amount of ε-PL into collagen self-assembly represents a promising approach for developing antimicrobial biomimetic collagen fibrils.