Methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii are important opportunistic bacteria responsible for serious hospital infections. These pathogens have become problems worldwide because of their high prevalence and resistance to a wide range of antibiotics. In this study, lethal and sublethal doses were estimated by intraperitoneal (IP) infection with A. baumannii and MRSA in C57-BL6 and Balb/c mice, respectively. Twenty-four hours post-infection, animals belonging to the groups that received doses of A. baumannii ATCC19606 greater than 2x105 CFU/mL were not able to survive; this was considered the lethal dose. However, some animals that received doses below 2x104 CFU/mL survived, allowing the LD50 for this bacterium to be established at approximately 1x105 CFU/mL. Meanwhile, groups that were infected with MRSA were able to survive at doses below 1.2x106 CFU/mL, which was found to be the LD50 in this trial. The Three Rs of humane animal experimentation were observed, allowing the reliable and rapid estimation of the lethal and sublethal doses in two models, using different mouse lineages and bacterial strains and employing four animals per group instead of 10, as recommended in the literature. We suggest that the same protocol could be used with other bacteria to determine lethal and sublethal doses, thus vaccine assays and other antibacterial strategies could be evaluated more quickly while using fewer animals.