This study aimed to establish the fingerprint of Physalis Calyx seu Fructus (PCF) and to study the relationship between spectrum and antibacterial activity.The fingerprint of 16 batches of samples was constructed by an ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high resolution mass spectrometry (UPLC-Q-Orbirap HRMS),then the cluster heat map and principal component analysis (PCA) were performed.In vitro antibacterial activities of different batches of PCF against Staphylococcus aureus and Escherichia coli were detected and then grey relational analysis was used to establish the spectrum-effect relationship.Thirty-eight common peaks were screened out from the fingerprint,including 22 sucrose fatty acid eaters,four physalins,luteoloside,luteolin and other compounds based on the accurate mass and mass fragmentation patterns,and combined with literature information and related reference materials.The results of cluster heat map analysis showed that PCF from different sources were clustered into three categories,which agreed with the result of PCA.All samples had different degrees of antibacterial activities.Grey relational analysis indicated that peak 27 had great contribution to antibacterial activities against S. aureus and peak 37,29,30,19,33,35,31,17,32,20 and 28 had great contribution to antibacterial activities against E. coli.Above all,all characteristic peaks were sucrose fatty acid esters,which indicated that sucrose fatty acid esters may be potential effective substances. The characteristic peaks which greatly contributed to the pharmacodynamics of PCF were screened out by establishing its fingerprint and spectrum-effect relationship,which provided the theorybasis for further study of the pharmacodynamics and pharmacology of PCF.