Ten catalpalactone derivatives were designed, synthesized, and their structures were identified by ¹H NMR, ¹³C NMR, and HRMS. All the analogs were evaluated for antimicrobial, cytotoxic activities and insecticidal activities. Compound g bearing α,β-unsaturated six-member lactone ring showed excellent inhibitory effect on the tested pathogens. Compounds d and g showed potential antibacterial activities against Escherichia coli, Staphyloccocus aureus and Micrococcus luteus, while compounds g and catalpalactone showed potent cytotoxic activities. Compounds d, g, h-4 and catalpalactone exhibited moderate lethal activity against brine shrimp. We propose that α,β-unsaturated six-membered lactone ring is the essential structure for antimicrobial, cytotoxic activities and insecticidal activity. It is the first time that the antimicrobial activity of catalpalactone and analogs has been reported. The pharmacokinetics of catalpalactone and g in Wistar rats were carried out. The results provide valuable references for the further study of catalpalactone, especially for the drug development and structure optimization.