AFhPs aggregated into rigid amyloid fibers, which agglutinated microbes and exerted as antibiofilm agents. AFhPs have no microbicidal activity and little or no cytotoxicity. AFhPs may be utilized as novel antibiofilm agents. Summary Evidence suggests that short amyloid‐forming peptides derived from bacterial proteomes have functional roles; however, the reported activities are diverse and the underlying mechanisms remain unclear. In this study, we simulated short amyloid‐forming peptides from the amyloid‐forming truncated protein C123 of Streptococcus mutans (S. mutans), studied their biological functions in microbial proliferation and biofilm formation, and further investigated the underlying mechanism. Fourteen hexapeptides were simulated, 13 of which were successfully synthesized. We found that the amyloid‐forming hexapeptides (AFhPs) displayed efficient broad‐spectrum antibiofilm activity against the Gram‐positive bacteria S. mutans, Streptococcus sanguis and Staphylococcus aureus, Gram‐negative bacteria Escherichia coli and fungus Candida albicans, by aggregating into rigid amyloid fibres agglutinating microbes, whereas the non‐amyloid‐forming hexapeptides (non‐AFhPs) did not. The AFhPs did not kill microbes and showed little or no cytotoxicity. Furthermore, a set of AFhPs displayed broad‐spectrum antibiofilm activity, regardless of its source. The microbial cell wall carbohydrates, peptidoglycan (PGN), lipoteichoic acid (LTA), glucan and zymosan A, mediated AFhP binding and triggered significant AFhP fibrillation. Although amyloid fibres agglutinated lipid membrane model – large unilamellar vesicles (LUVs) – and LUVs facilitated AFhP fibrillation, the roles of lipid membranes in AFhP antibiofilm activities remain to be elucidated. We highlight the potential use of AFhPs as novel antibiofilm agents.