Refractive index detector is usually used in the analysis of sugars in mushrooms, which is characterized by poor sensitivity, reproducibility, and susceptibility to interference from co-eluting sample components. In the current study, identification and determination of free sugars in mushroom samples by high-performance liquid chromatography coupled to corona charged aerosol detector (HPLC-CAD) were presented for the first time. The best chromatographic separation was performed on a Shodex Asahipak NH2P-50 4E 5 μm and mobile phase composed of 75% acetonitrile and 25% water with flow rate was 1 mL/min. The developed method offers good linearity in concentration range 0.001–0.01 or 0.01–0.2 mg/mL for tested compounds with R2 > 0.99. Limit of detection (LOD) for analytes was in the range of 7.1–120.2 ng on column. HPLC-CAD method showed very good reproducibility (RSD < 5.1%). Fructose, mannitol, and glucose were detected in all examined mushroom samples. For white Agaricus bisporus, mannitol was the most abundant sugar (7.575 mg/g dw), whereas trehalose for Pleurotus ostreatus (3.426 mg/g dw). The developed method was successfully applied for quantification of free sugars and mannitol in mushrooms. The optimized method proved to be sensitive, reproducible, and accurate.