Phytochemical investigation of Diospyros mespiliformis leaves resulted in the isolation of new acylated flavone isoscutellarein 7- O -(4′′′- O -acetyl)- β -allopyranosyl(1′′′ → 2′′)- β -glucopyranoside ( 1 ), along with eight known flavonoid metabolites, luteolin 3′,4′,6,8-tetramethyl ether ( 2 ), luteolin 4′- O - β -neohesperidoside ( 3 ), luteolin 7- O - β -glucoside ( 4 ), luteolin ( 5 ), quercetin ( 6 ), quercetin 3- O - β -glucoside ( 7 ), quercetin 3- O - α -rhamnoside ( 8 ), and rutin ( 9 ). Their structures were identified by analysis of spectroscopic (UV, NMR, and MS) data, as well as by acid hydrolysis for the isolated glycosides. The antioxidant activity of D. mespiliformis metabolites was determined by the DPPH radical-scavenging assay. The new acylated flavone ( 1 ) and flavonol O -rhamnoside ( 8 ) displayed the highest antioxidant activities with IC 50 values 15.46 and 12.32 μg/mL, respectively, with respect to the antioxidant ascorbic acid (IC 50 value 10.62 μg/mL). In addition, the isolated flavonoids were evaluated against four human pathogenic bacteria where the methylated flavone ( 2 ) exhibited potent activity against Escherichia coli with inhibition zone 34 mm, and mild activity of flavonol O -rhamnoside ( 8 ) against Staphylococcus aureus with MIC value 9.77 μg/mL. According to the MBC/MIC ratio, the antibacterial activity of the isolated flavonoids was considered flavonoid 2 is bactericidal nature against S. aureus, and flavonoids 3 and 4 are bactericidal against E. coli.