The resistance evolution of Spodoptera frugiperda (J.E. Smith) to insecticides and Bt proteins along with the intensive crop production systems adopted in Brazil make it challenging to implement integrated pest management. The adoption of alternative methods to manage pests is fundamental to the implementation of favorable integrated pest management and insect resistance management. Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a valuable tool for S. frugiperda control. The characterization of the baseline susceptibility of S. frugiperda populations and cross-resistance involving SfMNPV and major insecticides and Bt proteins have not yet been conducted. The objective of this study was to characterize the baseline susceptibility of S. frugiperda populations from five Brazilian States to SfMNPV (Cartugen, AgBiTech, Fort Worth,TX). Possible cross-resistance to insecticides and Bt proteins among resistant S. frugiperda strains was also assessed. There were no differences in the susceptibility of the studied populations to SfMNPV. The estimated diagnostic concentration may be utilized in future monitoring studies to SfMNPV. The SfMNPV presented no cross-resistance to the chemical insecticides and to the Bt proteins tested. Our results provide evidence of the biological activity and high potential of SfMNPV as a distinct insecticidal mode of action for use in rotation with other tools.This biological insecticide is known to have a favorable toxicological and ecotoxicological profile and will be a valuable tool in insect resistance management and integrated pest management programs for control of S. frugiperda.