ABSTRACT The aim of the present study was to characterize the probiotic potential of Lacticaseibacillus casei 4 N‐6 strain isolated from cow's milk. For this purpose, acid, bile salt, pancreatin, pepsin, phenol, and lysozyme tolerance, co‐aggregation and auto‐aggregation properties, phytase production, antibiotic resistance, and antibacterial properties were analyzed in vitro. The strain had relatively poor acid, bile salt, and pepsin tolerance. However, the strain showed a high pancreatin, lysozyme, and phenol tolerance. In addition, it exhibited moderate co‐aggregation with E. coli and good autoaggregation. Furthermore, the cell‐free supernatant of Lb . casei 4 N‐6 showed a high antimicrobial activity against Bacillus cereus (N32), Salmonella enteritidis (RK‐485), and Enterococcus faecalis (RK‐487). 4 N‐6 was resistant to vancomycin, teicoplanin, gentamicin, and ceflazidime. The 4 N‐6 strain did not show hemolytic activity. In addition, this strain was found to be able to produce phytase. All the findings obtained indicate that Lb . casei 4 N‐6 is promising as a potential probiotic candidate and has superior properties that can be evaluated as a probiotic in the future. However, further research and in vivo studies are needed to fully understand its mechanism of action and optimize its use as a probiotic.