Abstract Lactobacillus parafarraginis ZH1 is a novel anti-yeast lactic acid bacteria strain isolated by our group. However, its inhibitive activity against yeasts under different conditions of the medium was unknown. The present study was to clarify the suitable culture conditions for L. parafarraginis ZH1 producing an anti-yeast substance, hexadecanoic acid, and its inhibitive effect on pathogenic (Candida krusei 1045) and spoilage (Pichia membranefaciens 661) yeasts at the culture condition of high hexadecanoic acid production. Carbon sources, electron acceptors, pH, temperature, incubation time, and shaking speed influenced hexadecanoic acid production by L. parafarraginis ZH1. When the Rogosa and Sharp broth (MRS) medium was modified with fructose replacing glucose and with the supplement of 5 mol/L citric acid, and the modified MRS medium was stationarily cultured at initial pH 5.5 and 30 °C for 30 h, L. parafarraginis ZH1 produced the most hexadecanoic acid. L. parafarraginis ZH1 more effectively inhibited C. krusei 1045 and P. membranefaciens 661 in modified MRS medium at the above condition than in MRS, due to more hexadecanoic acid and acetic acid. This biotechnology method makes L. parafarraginis ZH1 capable of direct conversing fructose into anti-yeast hexadecanoic acid to extend the shelf life of feed and food, and to potentially reduce the threat of pathogenic yeasts to animals and humans.