Aim This work was conducted to produce, purify and characterize biosurfactants from Aspergillus flavus AF612 isolated from citrus fruit. Methods and results Biosurfactant named ‘Uzmaq’ was isolated from A. flavus AF612. The chemical characterization of the biosurfactant was conducted. Biosurfactant Uzmaq produced by A. flavus, was composed of methoxy phenyl oxime glycosides. Two molecular forms of the biosurfactant, Uzmaq‐A and Uzmaq‐B were isolated. Biological properties (antifungal activity) were evaluated. The fractions of the biosurfactant were isolated and their surface properties were analysed. Uzmaq‐A and Uzmaq‐B had critical micelle concentration (CMC) around 170 and 80 mg l−1, and lowered surface tension of water up to 20 and 25 m Nm−1 respectively. The biosurfactants were stable at pH 3–12 and temperature up to 80°C. Growth and biosurfactant production kinetics were also analysed. Conclusion Novel biosurfactant Uzmaq was produced from A. flavus, which was composed of methoxy phenyl oxime glycosides. The surface activity of Uzmaq was better than the maximum values of synthetic chemical surfactants. The biosurfactant showed antifungal activity and self‐assembling properties. Significance and Impact of the Study Aspergillus flavus AF612 can be used for commercial production of Uzmaq that may be employed for controlled drug release applications and bioremediation.