Neosartorya and Talaromyces are typical fungi capable of producing heat resistant ascospores responsible for the spoilage of processed fruit products. In this study, the heat activation rates of Neosartorya and Talaromyces ascospores were investigated in several suspending media at various heating temperatures. Ascospores were dispersed in pH 3.5 McIlvain buffer, organic acid/alcohol-supplemented McIlvain buffer and grape juice (pH 3.5, 5.0°Brix) prior to heat treatments. In McIlvain buffer, the number of germinating ascospores increased logarithmically with longer exposure to heating at all test temperatures. Heat activation rates (k values) accelerated with increasing temperature. The calculated activation energy (Ea) values were similar among ascospores from the same genus, but the Ea of the test Neosartorya spp. were greater than that of the test Talaromyces spp. Greater k values were calculated from acetate-supplemented McIlvain buffer and grape juice. Similarly, normal- and branched-chain fatty acids were shown to enhance the heat activation rate of the ascospores in McIlvain buffer systems. These results could assist the food industry in designing adequate thermal processes for food products against the heat resistant fungi.