Macrocybe gigantea is an edible wild mushroom with potential nutritional and bioactive value. The present study aimed to isolate, identify and optimize the cultivation parameters of M. gigantea and to evaluate its nutritional composition, phytochemical constituents and in-vitro antioxidant activity. Methods: Mushroom was identified using morphological characteristics and ITS rDNA partial sequencing followed by BLAST analysis. Growth media, pH, temperature and different lignocellulosic substrates were evaluated for mycelial growth and yield. Proximate composition (dry weight basis), phytochemical screening and antioxidant activity were analysed using standard methods including DPPH, FRAP and reducing power assays. Results: The collected mushroom was identified as Macrocybe gigantea based on microscopic examination and ITS rDNA sequencing, which showed 99.12% similarity (93% query coverage; E-value 0.0; max score 1112) in BLAST analysis. Paddy straw showed the highest biological efficiency (84.7±1.1%) compared with other substrates. Nutritional composition analysis on a dry weight basis revealed a high protein content (47.34±3.28%), followed by a substantial proportion of carbohydrates (37.21±1.86%), and a comparatively low fat content (3.04±0.24%). Additionally, it is a valuable source of vitamins B, C, and D. Phytochemical screening revealed the presence of phenolics, flavonoids and other bioactive compounds. The mushroom ethanol extract exhibited better antioxidant activity with an IC50 value of DPPH (57.07±1.71 μg/ml), ABTS (39.18 μg/ml±1.18) and phosphomolybdenum assay (IC₅₀: 45.22±1.36 μg/ml). Conclusion: Macrocybe gigantea demonstrated high biological efficiency on paddy straw and exhibited appreciable nutritional value along with significant in-vitro antioxidant activity, particularly in the ethanol extract. Further in-vivo studies are required to validate its potential health benefits.