Abstract Background Surveillance cultures are routinely used by public health authorities to screen for multidrug-resistant bacteria. Recently, the overuse of antibiotics intensified by the COVID-19 pandemic worldwide brought concerns about potential impacts on the acquisition and/or selective pressure of resistance genes, that in general carried by plasmids, which contribute to the rapid resistance spread within specific bacterial populations. The purpose of this study was to determine the prevalence of multidrug-resistant (MDR) organisms recovered from surveillance cultures (rectal, anal, nasal, and axillary) isolated from several Brazilian public hospitals. Methods Surveillance samples were analyzed from 1st January to 31st December 2023. All the swab samples were collected from patients admitted from high-risk settings or transferred from areas with high rates of MDR organisms. All clinical specimens were inoculated onto a selective media (ChromID media, bioMerieux) and incubated at 35±2°C for 18-24 h. The screening test was performed to Gram-negative bacilli (GNB) using the antibiotics ertapenem, imipenem and meropenem and for Gram-positive Cocci (GPC) screening was tested oxacillin (methicillin) and vancomycin using disc diffusion method. Bacteria identification was performed by MALDI-TOF mass spectrometry (Vitek-MS and bioMerieux) and as required, the minimal inhibitory concentrations (MIC) of antibiotics were determined using the Vitek 2 System (bioMerieux). Results A total of 71,064 rectal swab samples were processed. 4,652/71,064 (6.5%) samples showed positive results for the presence of 5.255 MDR microorganisms. Out of which, 4,477/5,255 (85.2%) were Gram-negative Bacilli (GNB) and 778/5,255 (14.8%) were Gram-positive Cocci (GPC). The majority of the isolated carbapenems resistance was Klebsiella spp. [2,791/5,255 (53.1%)], which the Klebsiella pneumoniae was the predominant species, followed by Acinetobacter baumannii [1,268/5,255 (24.1%)], Pseudomonas aeruginosa [263/5,255 (5%)], Enterobacter spp. [96/5255 (1.8%)], Serratia spp. [31/5,255 (0.6%)], Citrobacter spp. [22/5,255 (0.4%)], and others BGN [6/5,255 (0.2%)]. The resistance to vancomycin and methicillin were observed in 10% (525/5,255) of Enterococcus spp. and 4.8% (253/5,255) of Staphylococcus aureus, respectively. Conclusions The study found that K. pneumoniae carbapenem-resistant and Enterococcus spp. vancomycin-resistant were the main Gram-negative and Gram-positive organisms recovered from surveillance culture samples. However, our data draws attention to the increased cases of carbapenem-resistant Acinetobacter baumannii as the second most frequent pathogen isolated from surveillance samples after the last wave of COVID-19 pandemic. Active surveillance culture for target microorganisms followed by contact precautions to minimize the chance of transmitting MDR organisms from colonized to non-colonized patients consist of an important activity of infection control policy.