To improve the biological control agent (BCA) efficacy, stress factors threatening the microorganisms viability during spray application need to be determined. The effect of spray mixture temperature and exposure time on Trichoderma harzianum T 22 and Bacillus amyloliquefaciens QST713 viability were tested. Concurrently the combined effect of mechanical and thermal stress effect on BCAs viability were tested at two initial spray mixture temperatures (14°C and 25°C) by simulating a spray application using airblast sprayers featured by different tank capacity and a spray liquid circuit (without and with hydraulic agitation system). To assess the BCA microorganisms viability, spray mixture samples were collected at time intervals along trials and plated to count the colony forming units (CFU). The critical temperature threshold that inhibited BCA viability was 35°C with 30 min of exposure. The sprayer type, the initial temperature of the spray mixture and the temperature increment during the trials significantly decreased the number of the CFU recovered. When simulating a spray application, the spray mixture temperature increasing rate was mainly determined by the residual amount of spray mixture in the tank. Even if the tank capacity does not substantially affect the final temperature reached by the spray mixture, the higher residual spray mixture in bigger tanks can expose the BCA for a longer time to critical temperatures. Experimental trials allowed to identify the effect of factors affecting the viability of tested BCA, providing information about the actual chance to guarantee the biological efficacy of BCA treatments. This article is protected by copyright. All rights reserved.