Mushrooms provide essential nutrients and bioactive compounds, namely glucans, protein, and phenolic compounds. In this study, two aqueous extractions were performed (M1 and M2) using Pleurotus ostreatus, Pleurotus eryngii, and Agrocybe cylindracea. In M1, a hot extraction (extract M1) (90 °C, 700 rpm, 1 h) was performed. In M2, a room-temperature extraction (extract M2A) followed by a hot extraction (extract M2B) (90 °C, 700 rpm, 1 h) of the extract M2A residue was performed. The M2B extracts showed the lowest extraction yields (12.58–21.78%), while the other yields ranged between 30.91% and 46.03%. All extracts had high protein (12.09–32.97 g/100 g of dry extract), glucan (12.69–48.57 g/100 g of dry extract), and phenolic contents (7.90–16.65 mg GAEs/g of dry extract) and high antioxidant (ABTS and ORAC assays), antimicrobial, antibiofilm, and prebiotic activities. So, they have potential to be used as functional ingredients or natural preservatives. Extracts from A. cylindracea stood out since they had higher protein content, antioxidant activity, and prebiotic activity (extract M1) and inhibited a higher number of foodborne bacteria (only extract M2A). However, unlike extracts from P. ostreatus and P. eryngii, at 40 mg/mL, they had cytotoxic effects.