Aims: The cultures of an endophytic fungus Calcarisporium sp. were screened for inhibitors on the growth of feed-associated moulds and on the aflatoxin biosynthesis to find a safe and effective feed preservative. Methods and Results: Eight test fungi were isolated from the spoiled poultry feed. The endophytic fungus Calcarisporium sp. was separated from the Chinese coastal grass Cynodon dactylon. The antifungal action concerning the endophytic culture extract (ECE) was performed with propionic acid (PPA) as the corresponding reference. The ECE had a similar antifungal efficacy to PPA in a concentration-dependent manner. The susceptibility order of the ECE to the test fungi was found to be Fusarium sp. > Aspergillus spp. > Penicillium spp. Furthermore, the application of the ECE in pelleted-layer duck feed as a preservative was carried out at a humidity of 10, 15 and 20%. It has been discerned that mould growth and aflatoxin biosynthesis could be co-inhibited almost completely by ECE at concentrations higher than 1.0% (w/w). The LD50 of the ECE on mice was shown to be higher than 28 g kg(-1). Conclusions: The ECE can be selected as an inhibitor to preserve poultry feed on inhibiting the growth of mould and aflatoxin biosynthesis during feed storage. Significance and Impact of the Study: The ECE may be an effective and biosafe antifungal ingredient for poultry feed and holds a potential market prospect in feed industry.