Research Article Identification and Antibacterial Characterization of Endophytic Fungi from Artemisia sieberi Ibtisam Mohammed Ababutain , 1,2 Sahar Khamees Aldosary, 1,2 Amal Abdulaziz Aljuraifani, 1,2 Azzah Ibrahim Alghamdi, 1,2 Amira Hassan Alabdalall, 1,2 Eida Marshid Al-Khaldi, 1,2 Sumayh A. Aldakeel, 3 Noor B. Almandil, 4 Sayed AbdulAzeez, 3 and J. Francis Borgio 3,5 1 Department of Biology, College of Science, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia 2 Basic & Applied Scientific Research Center (BASRC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia 3 Department of Genetic Research, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia 4 Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia 5 Department of Epidemic Diseases Research, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Correspondence should be addressed to Ibtisam Mohammed Ababutain; iababutain@iau.edu.sa Received 24 December 2020; Revised 2 February 2021; Accepted 22 February 2021; Published 8 March 2021 Academic Editor: Elena Sorrentino Copyright © 2021 Ibtisam Mohammed Ababutain et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Endophytic fungi serve as a reservoir for important secondary metabolites. e current study focused on the antibacterial properties of endophytic fungi isolated from Artemisia sieberi. Initially, six endophytic fungi were isolated and purified from the stem of A. sieberi. Endophytic fungi were identified by morphological characteristics, as well as by molecular identification using 18S rRNA gene sequencing method. All the six isolates were subjected to the preliminary screening for their antibacterial activity against nine important pathogenic bacteria using the disk-diffusion method. Crude extracts of the most active isolate were obtained using ethyl acetate. Antibacterial activity of the ethyl acetate extract was evaluated using well diffusion method on the selected isolate. e antibacterial efficiency of the selected isolate was evaluated by determining the Minimum Inhibitory Concentration (MIC). MIC values were in appreciable quantity against both Gram-positive and Gram-negative bacteria ranging from 3.125 to 6.25 µg/mL and 12.5 to 50 µg/mL, respectively. is result indicated that Gram-positive bacteria were more susceptible to the endophytic fungi extract. Moreover, the molecular identification results revealed that all the isolates belong to Ascomycota and represented Aspergillus and Penicillium genera and three species: A. oryzae (three isolates), A.niger (one isolate), and P. chrysogenum (two isolates). All six endophytic fungi were able to inhibit the growth of at least two of the tested bacteria. Among the isolated strains, isolate AS2, which identified as P. chrysogenum, exhibited the highest antibacterial activity against all nine tested bacteria and was higher than or equal to the positive control against most of the tested bacteria. Future studies are required to isolate and identify these bioactive substances, which can be considered as a potential source for the synthesis of new antibacterial drugs to treat infectious diseases. Hindawi International Journal of Microbiology Volume 2021, Article ID 6651020, 11 pages https://doi.org/10.1155/2021/6651020