BACKGROUNDIn the present study, the optimization of ultrasound‐assisted alkaline extraction (UAAE) and isoelectric precipitation (IEP) was applied to white button (WBM) and oyster (OYM) mushroom flours to produce functional spray dried mushroom protein concentrates. Solid‐to‐liquid ratio (5–15% w/v), ultrasound power (0–900 W) and type of acid [HCl or acetic acid (AcOH)] were evaluated for their effect on the extraction and protein yields from mushroom flours submitted to UAAE‐IEP protein extraction.RESULTSPrioritized conditions with maximized protein yield (5% w/v, 900 W, AcOH, for WBM; 5% w/v, 900 W, HCl for OYM) were used to produce spray dried protein concentrates from white button (WBM‐PC) and oyster (OYM‐PC) mushrooms with high solids recovery (62.3–65.8%). WBM‐PC and OYM‐PC had high protein content (5.19–5.81 g kg−1), in addition to remarkable foaming capacity (82.5–235.0%) and foam stability (7.0–162.5%), as well as antioxidant phenolics. Highly pH‐dependent behavior was observed for solubility (> 90%, at pH 10) and emulsifying properties (emulsification activity index: > 50 m2 g−1, emulsion stability index: > 65%, at pH 10). UAAE‐IEP followed by spray drying increased surface hydrophobicity and free sulfhydryl groups by up to 196.5% and 117.5%, respectively, which improved oil holding capacity (359.9–421.0%) and least gelation concentration (6.0–8.0%) of spray dried mushroom protein concentrates.CONCLUSIONOverall, the present study showed that optimized UAAE‐IEP coupled with spray drying is an efficient strategy to produce novel mushroom protein concentrates with enhanced functional attributes for multiple food applications. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.