The rising concern over food safety and spoilage caused by fungal contamination has highlighted the need for natural and effective bio-preservatives. This study aimed to isolate and identify lactic acid bacteria (LAB) from dairy products with antifungal properties and to characterize their bioactive compounds for potential use as bio-preservatives. 208 isolates were isolated from the dairy products, from 80 LAB isolates, only four demonstrated the ability to inhibit the growth of Aspergillus flavus. The scope of the study was then expanded to assess the antifungal effects of these isolates against a broader range of fungal strains, including Aspergillus niger, Mucor sp., Rhizopus stolonifer, Alternaria citri, Aspergillus niger ch01, and Aspergillus flavus ch02. LAB isolate “B 50" exhibited the strongest antifungal activity, effectively inhibiting both fungal spore germination and mycelial growth. Molecular identification confirmed “B 50" as Lacticaseibacillus rhamnosus. The shelf life of cheese slices with added supernatant of L. rhamnosus were longer than control. The cell-free supernatants of selected L. rhamnosus was identified and quantified as formic, lactic, acetic and succinic acids at concentration of 1.541, 18.535, 7.113 and 7.154 mg ml-1, respectively. The integration of both in vitro and in situ screening experiments allowed to selection the high significant strain L. rhamnosus target fungal growths of selected fungi, with antifungal activities as food bio-preservatives.