This study focuses on the isolation of probiotics from Deutoplasm and characterization of the probiotic potential of the isolates. Probiotics are live microorganisms that aid in maintaining our gut in healthy condition. In this study, Lactobacillus sp. were isolated from deutoplasm (boiled egg yolk) and identified based on the morphologies of their colonies and biochemical assays. Among 15 isolated LAB species, 5 were obtained after identification and purification. The five isolates (A, B, C, D, E) were further analyzed for their probiotic characteristics – acid tolerance, bile tolerance and auto-aggregation capacity. The antimicrobial activity was assessed and isolate B was found to be more resistant to the pathogens Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumonia, and E. coli. Additionally, bile salt hydrolase activity and antibiotic resistance were examined for safety assessment. The isolates A and B were found to be highly resistant to the antibiotics – Vancomycin and Gentamicin. These five Lactobacillus isolates from boiled egg yolk are therefore intriguing candidates to be added to functional fermented foods as probiotics.