The combined effect of pH (3.5-7.0), temperature (4°C-50°C), and the presence of nisin (0-200 μg/mL) on the inactivation caused by pulsed electric fields (PEF) in two PEF-resistant Gram-positive microorganisms, Staphylococcus aureus and Listeria monocytogenes, was investigated. A response surface model using a central composite design was developed for the purpose of understanding the individual effects and interactions of these factors and to identify the most promising combinations for microbial inactivation. According to the developed models, temperature was the factor showing the greatest influence on PEF inactivation in the two strains investigated. A temperature increment from 4°C to 50°C increased the lethality of PEF by 2 and 3 log(10) cycles in S. aureus and L. monocytogenes, respectively. PEF inactivation in both microorganisms decreased with increased pH in the treatment medium from 3.5 to 7. The effect of the presence of nisin on the increment of PEF lethality for L. monocytogenes was additive or slightly synergistic. For S. aureus, this effect was synergistic at low temperatures and tended to disappear with increasing temperature. An inactivation of 4.5 and 5.5 log(10) cycles was achieved in the populations of S. aureus and L. monocytogenes, respectively, in a medium of pH 3.5 in the presence of 200 μg/mL of nisin at 50°C. Therefore, the application of PEF at moderate temperatures in the presence of antimicrobials such as nisin has great potential for achieving effective control of the vegetative forms of the two PEF-resistant Gram-positive strains investigated, especially in foods of low pH, such as fruit juices.