Biosurfactants (BS) are surface-active agents derived from microbes, garnering increasing industrial interest due to their environmentally friendly and sustainable nature. Although several bacterial isolates capable of producing biosurfactants have been reported, their production by diazotrophic strains remains underexplored. This study investigated the production, physicochemical properties, chemical composition and antifungal activity of BS produced by the diazotrophic bacteria Paenibacillus sp. ESA 664, isolated from the semi-arid region of Brazil. The results showed that the isolate was able to grow and produce BS in mineral media using glucose and ammonium chloride as carbon and nitrogen sources, respectively. The chemical structure of the surfactant was elucidated using liquid chromatography-mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Results revealed a lipopeptide structure similar to surfactin with the C15 isoform being the most abundant. The surfactin obtained showed surface tension of 24.4 mN/m, critical micellar concentration (CMC) of 3.88 mg/L, interfacial tension of 2.87 mN/m and emulsification index of 62.0%. In addition, the BS presented stability over a wide range of temperatures (-20 to121 degrees C), pH (2 to 12), NaCl (1 to 20%), and sucrose (1 to 5%), characterizing the product as a highly effective surfactant with potential for application in various industrial fields. Moreover, the compound exhibited significant fungicidal activity against the phytopathogen Lasiodiplodia theobromae, suggesting its use in agriculture as a biocontrol agent. Future investigations should address the development of field-applicable formulations and the elucidation of its antifungal mode of action.