The purpose of this work was to prepare and characterize an innovative formulation for vaginal delivery of econazole nitrate, commonly used for the treatment of Candida infections. A novel biosurfactant isolated from a vaginal Lactobacillus strain was used to prepare phosphatidylcholine based mixed vesicles. Biosurfactant was produced by Lactobacillus gasseri BC9, isolated from the vagina of a healthy premenopausal woman, and was chemically characterized by FT-IR and ESI-MS. Mixed vesicles, obtained through film rehydration and extrusion method, were characterized in terms of size, zeta potential, encapsulation efficiency, mucoadhesion properties and econazole release. Moreover, the antimicrobial activity of the mixed vesicles was tested towards both planktonic cultures and biofilms of Candida albicans. Biosurfactant produced by L. gasseri BC9 was composed by peptide-like molecules containing hydrocarbon chains and possessed a high surface activity together with a low critical micelle concentration. All the mixed vesicles presented optimal diameter range (226–337 nm) for topical vaginal administration. Econazole-loaded mixed vesicles containing biosurfactant showed higher encapsulation efficiency and mucoadhesion ability with respect to vesicles containing Tween 80. Further, they allowed a sustained release of econazole nitrate, maintaining the antifungal activity against C. albicans planktonic culture. Notably, biosurfactant-based vesicles were significantly more active than free econazole in the eradication of Candida biofilm. In conclusion, mixed vesicles are promising new vaginal delivery systems for the potential employment in the treatment of chronic infections.