Amauroderma rugosum (Blume and T. Nees) Torrend is a traditionally well-known mushroom that is used for the treatment of cancer. In order to evaluate the pharmacological activities of A. rugosum polysaccharides, the mushroom powder was subjected to hot water extraction and pure polysaccharides (ARPs) were isolated by gel-filtration method. Three important APRs called ARP-1, ARP-2 and ARP-5 were identified with average molecular weights of 1494, 450, and 7 kDa respectively. Their antioxidant abilities were estimated by examining free radical scavenging potential against 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid radical (ABTS●+), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH●), and hydroxyl radical. Immunomodulatory potentials of these ARPs were determined using murine macrophage cells. These polysaccharides exhibited high antioxidant abilities and stimulated mouse macrophages leading to the generation of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Excellent activities were displayed by ARP-1 and APR-2. Gas chromatography and spectroscopic (FT-IR and NMR) methods were employed in order to carry out their structural characterisation. The two high molecular weight ARPs (ARP-1 and ARP-2) displayed β-(1 → 3)-D-glucan backbone structure with branching of β-(1 → 6)-d-glucopyranosyl. These observations suggest high potential of ARPs for immunotherapeutic applications. •Five polysaccharides have been isolated from Amauroderma rugosum.•Amauroderma rugosum Polysaccharides exhibit significant immunostimulatory activities and low toxicity.•Two of the identified Amauroderma rugosum Polysaccharides have high potential to develop immunotherapeutic agents.•Two active polysaccharides (ARP-1 and ARP-2) exhibit β-(1. → 3)-D-glucan backbone structure with branching of β-(1 → 6)-d-glucopyranosyl