Extended from our previous finding that poly (3-hydroxybutyrate) (PHB) oligomer is an effective antimicrobial agent against gram-positive bacteria, gram-negative bacteria, fungi and multi-drug resistant bacteria, this work investigates the effect of polyethylene glycol (PEG) on the antimicrobial effect of PHB oligomer. To investigate and explain this promoting phenomenon, three hypothetic mechanisms were proposed, that is, generation of new antimicrobial components, degradation of PHB macromolecules and dissolution/dispersion of PHB oligomer by PEG. With a series of systematic experiments and characterizations of high-performance liquid chromatography–mass spectrometry (HPLC-MS), it was deducted that PEG promotes the antimicrobial effect of PHB oligomer synergistically through dissolution/dispersion, owing to its amphipathy, which improves the hydrophilicity of PHB oligomer.