In this study, we report ultrafine‐sized antibiotic dot particles targeting gram‐negative bacteria. The water‐soluble ultrafine‐sized hyaluronate dot (dHA) was conjugated with polymyxin B (PMB, as a target material for lipopolysaccharide on a gram‐negative bacteria) and chlorine e6 (Ce6, as a model photosensitizer), and it was denoted as dHA‐(PMB/Ce6). These dot particles interacted with lipopolysaccharide (LPS) on gram‐negative bacteria, which enabled PMB‐mediated docking to the outer membrane of gram‐negative cells. In the antimicrobial experiments using Escherichia coli cells, the phototoxic singlet oxygen generated from the dHA‐(PMB/Ce6) under light irradiation inhibited E. coli cell growth. This demonstrated that dHA‐(PMB/Ce6) can provide light‐activated selective phototoxicity against gram‐negative bacteria. We expect that the dot particle system can be used as an efficient method for improved antimicrobial treatment.