Novel refreshments with pleasant flavors were developed by fermentation of wort with basidiomycetes. Among 31 screened fungi, shiitake (Lentinula edodes) produced the most pleasant flavor. It was perceived as fruity, slightly sour, and plum-like. Flavor compounds were isolated by liquid-liquid extraction (LLE) and by headspace solid phase microextraction (HS-SPME). The key odor-active compounds were analyzed by a gas chromatography system equipped with a tandem mass spectrometry detector and an olfactory detection port (GC-MS/MS-O) and aroma extract dilution analysis (AEDA). For HS-SPME, a revised method of increasing the GC inlet split ratio was used. Most of the key odor-active compounds (e.g., 2-acetylpyrrole, β-damascenone, (E)-2-nonenal, and 2-phenylethanol) were detected with both extraction techniques. However, distinct differences between these two methods were observed.