Agaricus bisporus is one of the widely cultivated edible mushrooms and plays an important role in human nutrition. Therefore, revealing the biological and pharmacological properties of the mushroom will contribute to a more conscious approach towards its consumption. This study aimed to comprehensively investigate and compare the biological activities of extracts obtained from A. bisporus by Soxhlet ethanol and aqueous extraction (SEE and SEW, respectively) and ultrasonic-assisted ethanol and aqueous extraction (UAE and UAW). The phenolic composition was determined by HPLC analysis, among 21 standards; quercetin was found to be the most prominent. Different techniques were used to determine antioxidant activity, including phosphomolybden, radical scavenging (DPPH and ABTS), power reducing (FRAP and CUPRAC), and iron chelation experiments. The total phenolic content and total flavonoid content were tested. It was generally determined that ethanol extracts were rich in phenolic compounds, had higher total phenolic content, and exhibited higher antioxidant activity. Enzyme inhibitory activities were tested using different methods, including determination of acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, and tyrosinase activities. Significant enzyme inhibitory activity was seen in SEE against all enzymes. The antiviral activity of non-toxic extract concentrations against HRSV and HSV-1 viruses was investigated. All tested extracts demonstrated anti-HRSV activity, with the highest activity observed in the SEW extract [selective index (SI) = 135.90]. Anti-HSV-1 activity was detected only in UAW extract (SI = 85.86). According to the antiviral activity results, it was seen that the extracts showed higher anti-HRSV activity. These results emphasise the health benefits of A. bisporus as well as its role in nutrition.