Recently, research for alternative methods to combat gastrointestinal parasites has increased, and the biological control activity of the fungus Duddingtonia flagrans stands out. In this study, the possible influence of temperature on the nematophagous activity of D. flagrans, after gastrointestinal passage, against Haemonchus contortus in sheep was analysed. Four female sheep, between 2 and 3 years of age and weighing between 40 and 50 kg, were used. Two sheep were parasitised with H. contortus, while two other sheep were dewormed. Before the collection of faeces, one of the dewormed animals received a dosage of 1 × 106 chlamydospores of D. flagrans, lyophilised in gelatin capsules, for three consecutive days. The faeces were collected with collector bags, mixed, and then separated as samples with (fungus; 800 eggs per gram (EPG) of faeces) or without fungus (control; 900 EPG). Each sample (five replicates) was maintained in a biochemical oxygen demand incubator under different temperatures (5, 10, 15, 20, 25, 30, or 35 °C) for 21 days, followed by determination of the larval recovery. Compared to the control group, the best temperature for fungal action was 30 °C, while no larvae were recovered at 5 °C. At 10 °C, fungal action was detected, yet there was no significant difference in the percent larval reduction between all temperatures, demonstrating that larval presence seems to be the main factor affecting the nematophagous action of D. flagrans. Temperature does not appear to be a limiting factor in the biological control activity of D. flagrans against H. contortus, but larval presence, which was not observed at 5 °C, is mandatory. At low temperatures, which are typically suboptimal conditions for fungal and larval development, the lyophilised D. flagrans reduced the number of H. contortus larvae, which demonstrates the biological control potential and the potential use of D. flagrans in the subtropics.