Background: The marine environment is the main source of research on natural products in the future. In addition, marine microorganisms have been identified as a natural source capable of developing new antibiotic compounds, including controlling urinary tract infections caused by Escherichia coli ESBL. Purpose: This study aims to isolate, select, and test the potential of brown macroalgae endophytic fungi (Phaeophyceae) collected from the coastal waters of Sanur, Bali Province. Methods: Three types of brown algae were collected from Sanur Beach and their endophytic fungi were isolated using PDA media. Antibacterial activity was determined by measuring the inhibition zone and determining the inhibition category. The selected isolates were cultured and the phytochemical profile was determined qualitatively. In addition, molecular identification using the Internal Transcribed Spacer (ITS) primer set and comparison with the GenBank (NCBI) database were carried out in this study. Results: The results showed that 10 isolates were successfully isolated from three types of brown macroalgae. It was found that isolates T1, S1, and P3 had the highest inhibition in the very strong category. There were variations in mycelial weight, pH value, and inhibition shown by the culture filtrate of the three endophytic fungal isolates against Escherichia coli ESBL. Isolate S1 had the highest phytochemical profile, namely alkaloids, triterpenoids, saponins, and phenolics. The three endophytic fungal isolates showed isolate T1 (Phlebiopsis magnicystidiata) (MT5617191), isolate P3 (Neurospora crassa strain RT3M) (MT1028551), and isolate S1 (Peniophora sp.) (MH2680421). Conclusion: The results of this study provide initial information regarding the potential of bioprospecting brown macroalgal endophytic fungi as a source of new antibiotics against Escherichia coli ESBL.