In the era of antibiotic resistance, probiotics have emerged as potential therapeutic alternatives to antibiotics. The probiotic potential of Lactobacillus is well known, but it is also a promising source of novel pharmaceutical products and is used in various food applications such as functional foods, antibiofilm agents, and antimicrobials. Moreover, the synthesis of various chemicals, pharmaceuticals, organic acids, biosurfactants, and bacteriocins from these bacteria has been previously reported. Because of the increasing occurrence of antibiotic resistance, biofilm infections are remarkably difficult to treat. Furthermore, recent studies have acknowledged the role of biosurfactants released by Lactobacillus sp. in restricting the development of biofilms owing to their well-known activity as a biosurfactant in biomedical and food processing industries. Biosurfactants produced by Lactobacillus sp. have shown antimicrobial activity by interfering with the biofilm formation; therefore, it is important to understand the role of biosurfactants from Lactobacillus sp. as antibiofilm agents. In this review, we attempt to focus on the problems associated with the production of pathogenic biofilms in the biomedical sciences and food industry. Furthermore, the importance of biosurfactants from probiotic Lactobacillus sp. in inhibiting biofilm production has also been discussed.