This study was oriented to production of bioactive tetradecane reported in extracts of Pediococcus acidilactici isolated from curd milk and 16S rRNA sequence analysis of isolate showed 98% correlation with P. acidilactici DSM: 20284-CM (accession no. NR042057.1). P. acidilactici activity has been tested by disc diffusion assay against Staphylococcus aureus MRSA ATCC 43300, Listeria monocytogenes ATCC 19116, Pseudomonas aeruginosa MTCC 1934, and Escherichia coli MTCC 1610, Clostridium bifermentans MTCC 11273 and Candida albicans MTCC 183. The highest antibacterial and antifungal activities created by this strain were obtained. Tetradecane active metabolite which was extracted by ethyl acetate contained one observed bioactive spot on thin layer chromatography plate. Spectroscopic analyses confirm the presence of tetradecane as pure product. Minimal inhibitory concentrations (MIC) of tetradecane against indicators are noteworthy. In conclusion, this is the first work show tetradecane was obtained from a novel P. acidilactici-CM strain. Tetradecane act as antibacterial and antifungal.