The study aimed to assess the probiotic potential of Lactobacillus acidophilus BIOTECH 1900. The isolate was evaluated in vitro for a series of functional, safety and technological properties that could enable its use as potential probiotics. In terms of its physiological functionality, the isolate was able to tolerate pH 3.0 and 0.3% bile salts with a resistance rate of 56.43% and 89.00%, respectively. It displayed a strong antagonistic activity against Escherichia coli and Staphylococcus aureus. The isolate demonstrated a strong auto-aggregating phenotype with 84.40% and 87.97% activity after a 3- and 5-hour incubation, respectively. However, it exhibited a low cell surface hydrophobicity in xylene (3.51%). Considering its safety aspects, the isolate exhibited γ-hemolysis. It was susceptible or moderately susceptible to antibiotics including amoxicillin, ampicillin, augmentin, penicillin G, erythromycin, clindamycin, chloramphenicol and tetracycline. However, it was resistant to cefaclor, kanamycin and streptomycin. Regarding its technological properties, the isolate demonstrated tolerance up to 9% (w/v) NaCl. It also exhibited the same behavior with regard to lactic acid production and viable cell count in different milk systems (cow, buffalo and goat’s milk). The results show that L. acidophilus BIOTECH 1900 can be labeled as potential probiotics as it displayed desirable functional, safety and technological properties.