The mammalian gut microbiota plays a crucial role in promoting host health, and lactic acid bacteria (LAB) are commonly employed as probiotics for their beneficial effects. The Hangul deer (Cervus hanglu hanglu), a critically endangered red deer subspecies found in the Indian subcontinent, requires meticulous health management for its conservation. This pioneering study aimed to isolate, identify, and evaluate the in-vitro probiotic functional properties of LAB strains from the faeces of Hangul deer. A total of 27 LAB strains were isolated and identified using 16S rDNA gene sequencing, followed by comprehensive probiotic characterization and safety assessment. Remarkably, four species exhibited robust resistance and survivability against varying pH levels and bile salts, along with high aggregation and co-aggregation capacities. Notably, Lactobacillus acidophilus and Enterococcus mundtii strains displayed antibacterial activities. Safety assessment revealed the absence of hemolytic activity and virulence genes in all four strains. Antibiotic susceptibility testing showed that Lactobacillus acidophilus and Enterococcus casseliflavus were susceptible to all tested antibiotics, while Enterococcus mundtii exhibited resistance to clindamycin, and Enterococcus gallinarum exhibited resistance to erythromycin. These findings suggest that the isolated LAB strains possess advantageous probiotic characteristics and hold potential as dietary supplements for promoting the health and disease management of Hangul deer.