Resistance to antimicrobial agents is increasing worldwide and imposes significant life-threatening risks to several different populations, especially those in intensive care units (ICUs). Bacteria can quickly develop or acquire resistance to antimicrobial drugs, and combined with their intrinsic potential to cause disease in humans, these bacteria can become deadly. Among Gram-negative bacteria, Acinetobacter baumannii is notorious as a frequent opportunistic pathogen associated with critically ill patients, and understanding the genetic basis of A. baumannii resistance to beta-lactams among patients in ICUs will result in better protocols to prevent the development of resistance as well as improved treatment regimens. In this study, we assessed 1333 patients in five ICUs, 56 of whom developed A. baumannii infections. Most of the A. baumannii isolates were resistant to beta-lactam antimicrobial drugs, specifically, 3rd- and 4th-generation cephalosporins and carbapenems, and 91.1% of the isolates were multi-drug resistant (MDR). The most frequent OXA gene present was OXA-23 (55.1%), which is significantly associated with MDR strains. Most of the A. baumannii isolates (76.8%) were capable of forming a biofilm. The antimicrobial drug classes that were effective against most of these isolates were polymyxins and tigecycline. The molecular profile of the isolates allowed detection of 12 different clusters comprising 2 to 8 isolates each. In conclusion, our data indicate a high incidence of resistance to carbapenems as well as MDR strains among the observed A. baumannii isolates, most of which exhibited a high prevalence of OXA-23 gene expression. Only a few selective drugs were effective, reinforcing the notion that bacterial resistance is an emerging problem that should be prioritized in every healthcare facility.