The fruits of Cordia dichotoma G. Forst. (Boraginaceae) are commonly used as medicinal material to treat various ailments. In this paper, we performed bioassay-guided fractionation and isolation of bioactive constituents from the 80 % ethanol ultrasonic extracts of C. dichotoma fruits. The preliminary bioassay revealed that the ethyl acetate fraction (EA) of the extract exhibited stronger antioxidant: 2,2'-diphenyl-1-picrylhydrazyl (ABTS) with half inhibitory concentration (IC50) = 6.71 +/- 0.46 mu g/mL, 2,2'-diphenyl-1-picrylhydrazyl (DPPH) with IC50 = 34.18 +/- 0.52 mu g/mL, Ferric reducing antioxidant power (FRAP) with the value of 134.35 +/- 2.01 mu mol Fe2+/100 g) and antimicrobial (Candida albicans, Escherichia coli, and Staphylococcus aureus with the inhibition zones of 10.0, 9.0, and 12.0 mm, respectively) activities than other fractions. The EA fraction was further fractionated by silica gel column chromatography, and 45 compounds including 40 phenolics were isolated by various column chromatography and identified by spectroscopic data analysis. Among them, 41 compounds were isolated for the first time from C. dichotoma, including three previously undescribed phenolic derivatives. Most of the compounds showed significant antioxidant activity. Part of the individual compounds showed moderate or weak antimicrobial activity. And some compounds showed moderate antiviral activity (with IC50 = 12.84-115.56 mu g/mL) to influenza virus A/Hanfang/359/95(H3N2). Additionally, the structure-activity relationships of phenolic compounds were discussed systematically. Furthermore, the bioactivity contributions of individual compounds to fractions were discussed based on LC-MS and HPLC analysis. The results indicated that C. dichotoma fruits were a potential source of bioactive phenolic compounds, and the fruits extract could be applied as a preservative in food industry because of its potent antioxidant and antimicrobial activities.