Researchers reported studies on the compositions and biological activity of extracted oak galls. However, isolation and characterizing of bioactive compounds from the oak galls extracts have become challenges. In this study, a new bioactive compound was isolated and characterized from extracted locally oak goal crude (EOGC). A novel mixture of (methanol 10% + acetonitrile 10% +water 80%) solvents was performed to fractionate the soxhlet EOGC through a column chromatography technique. Thin Layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC) were utilized to study the constituents of the fractions. The isolated fractions were collected and tested against, Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 19404, Escherichia coli ATCC 8739, Salmonella ebony NTCT 6017 and Candida albicans ATCC 10231 utilizing the agar well diffusion method. Results demonstrated that only a single component fraction exhibited activity against (S. aureus, M. leutues, and C. albicans), in contrast, no detectable activity was observed against E. coli and S. ebony. Fourier Transform Infrared (FT-IR) and Mass Spectrometer techniques have been used to characterize the chemical composition of the compound. As a result, the new compound was postulated as Chrysin-gallate compound, which is considered a new bioactive chemical compound that may have a significant application as an antimicrobial and treat specific health issues.