Hop has been attracting scientific attention due to its favorable bioactivity properties. It is thus desirable to relate these properties to the specific hop compounds and extract these compounds in highly purified form in order to enhance the effect. The aim of the present study is the isolation of a sufficient amount of the highly purified prenylated minor hop compound xanthohumol C (XNC) for characterizing its bioactivity. Two strategies for the production of XNC were evaluated. The first strategy involved a capture of natural XNC from a xanthohumol (XN)-enriched hop extract (XF) by countercurrent chromatography. In the second approach, a one-step semi-synthesis of XNC was performed starting from XN, which had previously been separated from a natural XN-enriched hop extract. Both methods delivered XNC in sufficient amount and purity (>95%, HPLC), whereas the second strategy was preferable in terms of purity (>99%, HPLC) as well as productivity and solvent consumption. The methods were validated by identifying and quantifying XNC using LC-MS, LC-MS/MS and 1H NMR analysis. The XNC obtained in this way was supplied to several bacterial, yeast and fungal cultures in order to evaluate its antimicrobial effects. For comparison, microorganisms were also treated with the natural XN-enriched hop extract, as well as the prenylated hop compound XN. While still reducing cell proliferation, XNC was found to be less effective than both XF and XN for all studied bacteria and yeasts. Furthermore, for Bacillus subtilis, a strongly pH-dependent minimal inhibition concentration was observed for all three bioactive compounds, lowest at a pH of 5 and highest at a pH of 7.