•Composition of Diospyros virginiana methanol extract, phenolic profile.•Methanol extract, their isolated phenolic compounds have shown significant antimicrobial effect.•Flavonoid aglycones exhibited the strongest antimicrobial activity. This study was carried out with the objective to investigate antibacterial and antifungal activities of Diospyros virginiana fruits (methanol 80%) extract and the phenolic compounds isolated from the extract. The in vitro antibacterial and antifungal assays were done against eight bacterial strains and against eight fungal species using microdilution method. Eight phenolic compounds, m-gallate, gallic acid, luteolin, quercetin, myricetin, myricetin 3-O-α-rhamnoside, myricetin 3-O-β-glucoside, and myricetin 3-O-β-glucuronide were isolated and identified by different spectroscopic tools (UV, 1H NMR, 13C NMR, MS). The results of the methanol extract of Diospyros virginiana fruits and its isolated phenolic compounds showed significant antibacterial and antifungal effects against all the tested microorganisms. The best results were obtained for flavonoid aglycones which showed strong antimicrobial activity against all the microorganisms tested. These results may help to discover new chemical classes of natural antimicrobial substances that could serve as selective agents for infectious disease chemotherapy and control.