Chitin, a linear polymer of β-(1, 4) linked 2-acetamido-2-deoxy-D-glucopyranose, is the second most abundant biopolymer on earth next to cellulose and it is mostly obtained from the exoskeletons of arthropods such as crabs, shrimps, and prawns, and insects. Moreover, chitin can be also obtained from plant sources like mushrooms. Due to the versatile properties such as renewability, biodegradability, biocompatibility and non-toxicity, chitin and its derivative chitosan have many applications in different areas. Currently, industrial production of chitin and chitosan is from the shell wastes. However, we do not have such animal shell wastes that can be used us sources of the highly valuable biopolymers chitin and chitosan. Hence, the main objective of this study was to prepare partially deacetylated chitin nanofibers from locally available mushroom. The mushroom powder from the stem and cap was subjected to alkaline and acid treatments to remove proteins, soluble glucans, fats and minerals. The chitin content of mushroom in the stem and cap were estimated gravimetrically. The result clearly revealed that the stem of the mushroom has higher chitin content (16 wt. %) than the cap (7 wt. %). Chitin is insoluble in water and organic solvents due to the strong intra and inters molecular hydrogen bonds. To increase the process ability and functionality of chitin, it is usually subjected to deacetylation reaction, in which the acetyl group is changed into active amine groups. The degree of deacetylation is depending upon the concentration of the alkali, temperature and reaction time. In this study partially deacetylated chitin was synthesized using moderate alkali concentration; reaction time and temperature. This obtained chitin was disintegrated into partially chitin nanofibers with high performance commercial domestic blender. The synthesized Chitin nanofibers were characterized by UV-Vis, and FT-IR analyses. The synthesized partially chitin nanofibers were tested for antimicrobial activities against four human pathogens i.e E. coli, K.pneumonia, S. aureus and B. subtilis. They were found to have significant effect in controlling the growth of the human pathogens in which the chitin sample treated with 20 % alkali for deacetylation showed a maximum zone of inhibition, 18 mm in E.coli and a minimum of 8 mm in B.subtilis.