The Dayaknese consume Stenochlaena palustris for the treatment of various diseases. The plants contain chemical compounds that have the potential to be developed as antibiotics. This study aimed to isolate endophytic fungi from the leaves and stems of S. palustris, and further identify and assess the isolated endophytic fungi against microorganisms. The research was carried out in several stages, namely, isolation and purification of endophytic fungi, macroscopic and microscopic identification, activity test on isolates, fermentation and extraction of secondary metabolites, and evaluation of the antibacterial activity of the extracts using the disk diffusion method. This experimental laboratory research was conducted in Palangka Raya City, Central Kalimantan, with a test carried out at the Microbiology Laboratory of the Health Polytechnic Ministry of Health, Palangka Raya. The results showed 3 types of endophytic fungi: SpHtm, SpHjk, and Sporn, identified as Aspergillus sp., Paecilomyces sp., and Arthrocristula sp. The antimicrobial effect of the isolated fungi was assessed at various concentrations, i.e., 15, 30, and 45%, against S. aureus and E. coli. The results showed that the ethyl acetate extracts of the SpHtm and SpHjk isolates did not demonstrate any inhibitory response to S. aureus at concentrations of 15, 30, and 45% and did a small inhibitory response to E. coli at an SpHtm concentration of 30% and an SpHjk concentration of 45%, with inhibition zone diameters of 11.63 ± 0.01 and 10.83 ± 0.02 mm, respectively.