A novel series of 6-chloromethyl-3-hydroxy-2 substituted 4H-pyran-4-one derivatives were synthesized and tested for their antibacterial, antifungal and antiviral in vitro properties. In the view of activity results, compounds 8–11 (MIC: 8 lg/ml) were more remarkably active against Staphylococcus aureus and Enterococcus faecalis. Compounds 1–7 were highly active against Candida albicans and C. parapsilosis with MIC value of 8 lg/ml. Compound 9 bearing 3-chlorophenyl moiety was determined to be the most active compound against RNA virus PI-3.