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Introduction: The growing resistance to antibiotics and the complexity of defeating multi-drug resistant bacteria have led to an increase in the search for novel and effective antimicrobials from various plants. This study aimed to determine the bioactive contents of Auricularia auricular-judae mushroom and evaluate the antimicrobial potential of its protein extract against some selected human bacterial and fungal pathogens which could serve as a lead to the discovery of new antimicrobial agents., Methods: The constituents of the A. auricular-judae were evaluated by standard phytochemical analysis methods. The agar well diffusion, micro-broth dilution, and time-kill kinetic assays were used to determine the antimicrobial activity of the extracts against Staphylococcus aureus , Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa , Klebsiella pneumoniae , yeast ( Candida albicans ), and dermatophytic pathogens., Results: The preliminary phytochemical analysis of the extracts revealed the presence of carbohydrate (43.15 %; 38.30 %) proteins (23.75 %; 23.75 %), flavonoids (1.20 %; 0.80 %), alkaloids (0.60 %; 1.00 %), saponin (6.00 %; 2.40 %), tannin (1.65 %; 1.57 %), cyanide (0.24 %; 0.40 %), ash (12.40 %; 10.40 %), moisture (6.00 %;6.00 %), lipids(6.00 %;6.00 %), and fiber (8.70 %; 6.45 %) for the Tris buffer and warm aqueous extracts, respectively. The Tris and warm aqueous protein extracts showed antimicrobial effects toward all the human bacterial pathogens and two fungal isolates., Conclusions: This study revealed the potential ability of A. auricula-judae for use as a herbal antimicrobial in the treatment of human bacterial and fungal pathogens., Competing Interests: The authors declare that they have no conflict of interest., (© 2020 Elsevier GmbH. All rights reserved.)