Abstract: In order to explore the antibiotics development potential of the endophytic fungus Apiospora malaysiana from Thamnolia subuliformis, the antimicrobial spectrum of Apiospora malaysiana against common pathogen was determined after comparing the effects of six commonly used methods for determination of in vitro antimicrobial activity. The effects of storage time, temperature, ultraviolet irradiation time, and pH on the stability of antimicrobial activities of Apiospora malaysiana in vitro were further investigated by using the most sensitive Staphylococcus aureus as the indicator bacterium. The results showed that there was no significant difference in the antimicrobial activity of Apiospora malaysiana in vitro by three methods (plate punching method, filter paper method, and Oxford cup method) (P>0.05). However, the results of the three fungal inhabition methods showed that the mycelial growth rate method was optimal (inhibition rate of 100%), the effect of the plate confrontation method was much lower than the former (inhibition rate of only 24.0%), and the filter paper diffusion method had no inhibiting effect. The results of in vitro antimicrobial test showed that Apiospora malaysiana had strong inhibiting effects on a variety of Gram-positive bacteria and pathogenic fungi, among which Staphylococcus aureus was the most sensitive (the diameter of the inhibition zone was 18.3 mm, highly sensitive). In addition, Apiospora malaysiana also had strong antimicrobial activity against pathogenic fungi such as drug-resistant Candida albicans (highly sensitive), Botrytis cinerea, and Rhizoctonia solani (inhibition rate of 100%). The results of the antimicrobial stability test showed that different storage time, temperature, and ultraviolet irradiation time had no significant effects on the in vitro antimicrobial activity of Apiospora malaysiana (P>0.05), but pH had a great effect on its in vitro antimicrobial activity (P<0.05). When pH was lower than the background pH (5.5) of the extract, the diameter of the inhibition zone decreased. When pH was adjusted to higher than the background pH of the extract, the in vitro antimicrobial activity of Apiospora malaysiana disappeared. The results showed that the endophytic fungus Apiospora malaysiana from Thamnolia subuliformis had broad and great antimicrobial activity, and its antimicrobial stability in vitro was strong under normal conditions, which had the potential to be further developed into antibiotics and food preservatives.