Background: Research from developing countries, such as Nigeria, on Lactobacillus species in the female urogenital tract and their role as a barrier to vaginal infection is limited. Therefore, the aim of this study was to assess the clinical biotherapeutic potential of indigenous Lactobacillus species. Methods: Antimicrobial metabolites production were characterised using simple and easily reproducible qualitative and quantitative methods. The in vitro inhibitory effect of Lactobacillus antimicrobials on vulvovaginal candidiasis–associated Candida species was investigated using modified agar spot and agar well-diffusion methods. Results: The maximum levels of lactic acid, hydrogen peroxide, and diacetyl from 20 vaginal Lactobacillus strains from diseased subjects were 1.46 mg/L, 1.36 mmol/L, and 1.72 mg/L respectively. From the 4 healthy subjects, the maximum level of lactic acid was 1.08 mg/L; hydrogen peroxide, 1.36 mmol/L; and diacetyl, 0.86 mg/L. The maximum productions of these substances occurred between 72 and 120 hours of incubation. The in vitro antagonistic activities of vaginal L. acidophilus, L. fermentum, L. brevis, L. plantarum, L. casei, L. delbrueckii, and L. jensenii from diseased subjects inhibited a maximum of 5.71% of the 35 Candida species tested, while vaginal L. acidophilus and L. plantarum from healthy subjects inhibited between 57.1% and 68.6% of Candida species in vitro. Conclusion: Antimicrobial-producing lactobacilli can be considered as adjunct biotherapeutic candidates for the treatment of vulvovaginal candidiasis.