This study tested hyperbaric storage (25-150 MPa, for 30 days) at room-temperature (HS/RT, 18-23 °C) in order to control the development of Byssochlamys nivea ascospores in apple juice. In order to mimic commercially pasteurized juice contaminated with ascospores, thermal pasteurization (70 and 80 °C for 30 s) and nonthermal high pressure pasteurization (600 MPa for 3 min at 17 °C, HPP) took place, and the juice was afterwards placed under HS/RT conditions. Control samples were also placed in atmospheric pressure (AP) conditions at RT and were refrigerated (4 °C). The results showed that HS/RT, in samples without a pasteurization step and those pasteurized at 70 °C/30 s, was able to inhibit ascospore development, contrarily to samples at AP/RT and refrigeration. HS/RT for samples pasteurized at 80 °C/30 s evidenced ascospore inactivation, especially at 150 MPa, wherein an overall reduction of at least 4.73 log units of ascospores was observed to below detection limits (1.00 Log CFU/mL); meanwhile, for HPP samples, especially at 75 and 150 MPa, an overall reduction of 3 log units (to below quantification limits, 2.00 Log CFU/mL) was observed. Phase-contrast microscopy revealed that the ascospores do not complete the germination process under HS/RT, hence avoiding hyphae formation, which is important for food safety since mycotoxin development occurs only after hyphae formation. These findings suggest that HS/RT is a safe food preservation methodology, as it prevents ascospore development and inactivates them following commercial-like thermal or nonthermal HPP pasteurization, preventing mycotoxin production and enhancing ascospore inactivation.