High pressure processing is a mild preservation process that inactivates pathogenic and spoilage micro-organisms in food products, but preserves the fresh characteristics of a product. Compared to untreated product, an enhanced shelf life is obtained during refrigerated storage. Knowledge on the use of high pressure pasteurisation aimed for ambient storage is limited. The aim of this research was to investigate if a combination of high pressure and moderate heat could be used to produce a shelf-stable high-acid fruit product. Ascospores of the heat resistant fungi Talaromyces macrosporus and Aspergillus fischeri were added to fresh strawberry puree that served as a model system. The effect of the processing steps and storage at ambient temperature for 2 weeks was studied on viability of the ascospores. A preheating step at 69 °C/2 min resulted in full or partial activation of A. fischeri and T. macrosporus spores, respectively. The pressure build-up by the process without any holding time resulted in additional activation of spores. A combination of moderate heat (maximum 85–90 °C) and high pressure (500–700 MPa) for holding times up to 13 min inactivated these highly resistant spores much faster than a heat treatment alone. At Tmax = 85 °C and 600 MPa the spores of T. macrosporus and A. fischeri were inactivated by 5.0 and 5.5 log10 after 13 and 7 min, respectively. At Tmax = 85 °C the heat treatment alone did not reduce the viability of these spores up to 60 min of treatment. At Tmax = 90 °C the holding time of the combined pressure-heat treatment could be reduced to obtain the same degree of inactivation of the heat resistant fungi. In addition, treated and untreated ascospores in strawberry puree were stored for 14 days at room temperature to evaluate delayed outgrowth of spores. Untreated ascospores of A. fischeri were activated by storage in the puree. However, at conditions combining high pressure ≥ 600 MPa with Tmax ≥ 85 °C for 13 min, heat resistant fungi were successfully inactivated. This research showed that a combination of moderate heat and pressure can drastically improve the effectiveness to inactivate heat-resistant ascospores in a high-acid fruit product compared to a heat treatment, potentially resulting in a better product quality.