Fumonisins were first discovered in Fusarium verticillioides, a fungus associated to disease and asymptomatic infections in maize. Afterwards, other fungal taxa have been found to produce fumonisins. The entomopathogenic ascomycete Tolypocladium cylindrosporum has been isolated from soil and also as an endophyte from leaves of grasses. The objectives of this work were to determine the in vitro production of fumonisin B (FB) mycotoxins and the immunosuppressive compound cyclosporine A (CyA) in several strains of T. cylindrosporum, and to examine the effect of fungal virus infection and temperature in FB production. FB was detected in 30% of the strains, ranging from 0.16 to 5.52 μg cm in solid media, and FB was detected in 78% of the strains, ranging from 0.764 to 40.92 μg cm. CyA was not detected in any strain. The mean FB concentration of the endophytic strain Tc37W was three times greater (p < 0.05) than that of any other strain. Up to 34% more of FB was detected in strains infected by the virus TcV3 than in the corresponding virus-free versions. The effect of temperature on FB content was interactively significantly dependent on fungal strain and growth medium; in the YES medium, the FB of virus-infected strains Tc37-1V and Tc37W increased by 67 and 16%, respectively, at 26 °C as compared to 20 °C. The FB concentration in some fungal strains was similar to that in fungi associated to food and feed intoxications.