Lactiplantibacillus plantarum OV50 is a novel strain that was isolated from Algerian olives. Prior to its use as a natural biopreservative, OV50 underwent characterization for various functions. OV50 shows no proteolytic, lipolytic, or hemolytic activity. In addition, it is non-cytotoxic to eukaryotic cells and does not exhibit acquired antibiotic resistance. OV50 was tested with Pseudomonas aeruginosa ATCC 27835, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, and Vibrio cholerae ATCC 14035 in a sardine based-medium at 37 °C and 7 °C. At 37 °C, OV50 completely inhibited the growth of these foodborne pathogens for a maximum of 6 h. At 7 °C, it suppressed their growth for a maximum of 8 days, except for S. aureus ATCC 6538, whose growth was reduced from 4 to 2 log CFU/mL. Microbiological counts, total volatile basic nitrogen (TVB-N), and peroxide values (PV) concentrations were determined in fresh sardines inoculated with OV50 and kept at 7 °C for 12 days. The inoculated sardines showed a significant reduction in TVB-N levels at D8 (34.9 mg/100 g) compared to the control (59.73 mg/100 g) and in PV concentrations at D4 (6.67 meq/kg) compared to the control (11.44 meq/kg), as well as a significant reduction in the numbers of Enterobacterales, Coliforms, Pseudomonas spp., Vibrio spp., and S. aureus At D8 and D12 compared to the control. Taken together, these results indicate that OV50 can improve the microbiological safety, freshness, and quality of sardines.