It has been recently reported that, beta-Glucans with globular small particle size confer a higher disease resistance due to its induced strong immunomodulating activity. Herein, this work was designed to directly extract the nano beta-glucans (NGs) from mushrooms using an acid-base procedure. Two mushrooms, namely, Lentinula edodes (Shiitake) and Pleurotus ostreatus (oyster) mushrooms were used for the production of NGs-s and NGs-o, respectively, through boiling with 0.5 M NaOH for 24 h. The extracted NGs were then characterized using LC-MS, H-1-NMR, FTIR, SEM, TEM, DLS and UV-Visible spectra. Also, the antitumor activity of NGs was estimated against MCF-7 and HCT-116 cells. Results from LC-MS, proton NMR and FTIR as well as UV-vis analysis affirmed the successful extraction of NGs with the chemical structure of beta-glucan when compared with standard beta-glucan as a reference. In addition, morphological characteristics of NGs were obtained using SEM and TEM. SEM revealed the porosity of the surface with homogeneous distribution, particularly in the case of NGs-s. TEM also emphasized the formation of NGs of size ranging from 10 to 25 nm and 40-50 nm for NGs-s and NGs-o, respectively, with needle edge shapes as in NGs-s. However, NGs-o showed loosely aggregated irregular shapes which may be attributed to their low zeta potential. In vitro cytotoxicity assays affirmed the potential use of NGs as antitumor materials against carcinoma breast and colon cells at low doses compared to human normal Vero cells. This novel approach opens up opportunities for harnessing mushrooms in the preparation of new cost-effective bioactive products that can be applied in the medical sector.