A series of para-alkoxy phenyl-b-ketoaldehyde derivatives were synthesized and evaluated for antimicrobial activities. Preliminary results showed that some compounds had moderate antibacterial activities, especially, 3-(4-methoxyphenyl)-3-oxopropanal O-methyl oxime (4) showed more potent antibacterial activity against Staphylococcus aureus and resistant Helicobacter pylori than the other compounds. Compound 4 showed more active antibacterial activities against methicillin-resistant S. aureus than Houttuynin and levofloxacin. Compound 4 was also investigated and its antibacterial activities against the strains clinical separated of Staphylococcus aureus. The preliminary structure–activity relationships were obtained. The maximum tolerance dose value for acute toxicity of compound 4 was 5,000 mg/kg. It indicated that compound 4 should be safe in clinical treatment.