In 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. Prioritized 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. Overall, 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.