Background: One of the main objectives of the food industry is the shelf life extension of food products, taking into account the safety requirements and the preference of consumers attracted by a simple and clear label. Following this direction, many researchers look to find out antimicrobials from natural sources. Methods: Tomato, carrot, and melon by-products were used as substrates for lactic acid fermentation using seven strains belonging to the Lactobacillus genus, L. plantarum, L. casei, L. paracasei, and L. rhamnosus. The obtained fermented by-products were then extracted and the antimicrobial activity toward fourteen pathogenic strains of Listeria monocytogenes, Salmonella spp., Escherichia coli, Staphylococcus Aureus, and Bacillus cereus was tested through agar well diffusion assay. Results: All the extracts obtained after fermentation had highlighted antimicrobial activity against each pathogen tested. In particular, a more effective activity was observed against Salmonella spp., L. monocytogenes, S. aureus, and B. cereus, while a lower activity was observed against E. coli. Conclusion: Lactic acid fermentation of vegetable by-products can be a good strategy to obtain antimicrobials useful in food biopreservation.