Macrocyclic PKS-NRPS hybrid metabolites represent a unique family of natural products mainly from bacteria with broad and outstanding biological activities. However, their distribution in fungi has rarely been reported, and little has been reported regarding their nematocidal activity. Here we describe an unprecedented class of PKS-NRPS hybrid metabolites possessing a 13-membered lactam-bearing macrolactone, thermolides A-F (1-6) from a thermophilic fungus Talaromyces thermophilus. We showed that 1 and 2 displayed potent inhibitory activity against three notorious nematodes with LC(50) values of 0.5-1 μg/mL, as active as commercial avermectins. This work provided a new class of promising lead compounds for nematocide discovery.