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.

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