Carrot (Daucus carota L.) production is challenged by various phytopathogens, including Berkeleyomyces basicola, responsible for black root rot. Current control measures are limited, prompting interest in sustainable biopreservation approaches leveraging beneficial microorganisms. This study evaluated the biopreservation potential of several lactic acid bacteria (LAB) strains from the Agroscope Culture Collection and bacteria newly isolated from carrots against B. basicola and other carrot phytopathogens, namely Alternaria radicina, Rhizoctonia solani and Sclerotinia sclerotiorum. Results highlighted the superior performance of strains isolated from carrots, including Leuconostoc mesenteroides, Serratia plymuthica and Raoultella terrigena in inhibiting B. basicola mycelial growth and spore germination compared to the previously isolated LAB strains from the Agroscope Culture Collection. Interestingly, non-LAB strains, particularly Serratia plymuthica Sp1, exhibited broad-spectrum antifungal activity and sustained protection of carrots while used as postharvest treatment. These findings emphasize the value of exploring the microbiota of the host plant to be protected to find new agents suitable for biocontrol solutions. While LAB strains showed promising results in in vitro assays, S. plymuthica Sp1 emerged as a highly effective candidate for postharvest disease management. Future research should focus on optimizing the application and formulation of S. plymuthica for large-scale use, ensuring its compatibility with diverse carrot varieties and storage environments. This work contributes to the development of environmentally friendly strategies to reduce postharvest losses and enhance sustainability in food production.