Enzymatic systems of Aspergillus niger MB, Aspergillus niger KB and Penicillium chermesinum 113 catalysed hydroxylation at C-4’ in ring B of 5-hydroxyflavone and O methylation at the same position in 5-methoxyflavone. Additionally, in the culture of Aspergillus niger SBP 5-methoxyflavone underwent demethylation. An important factor was the time of substrate addition to the cultivation medium. The substrates were added either at the time of inoculation of the medium with a microorganism, or 24 h after the inoculation. For 5 hydroxyflavone better yields of transformation were observed when the substrate was added to the cultivation medium at the time of the inoculation, whereas, for 5 methoxyflavone the situation was opposite. Antioxidant properties of the substrates and the products of biotransformations were evaluated spectrophotometrically by DPPH assay.