Introduction: Increasing resistance to multiple classes of antimicrobials, leading to multidrug resistance (MDR), provides a major challenge for the management of bloodstream infections in the neonatal intensive care unit (NICU). The dissemination of Acinetobacter and Enterobacterales species were of great importance owing to their high prevalence and resistance against a broad spectrum of available antimicrobials, thereby exacerbating the prognosis for afflicted patients. This study aimed to highlight neonatal sepsis cases caused by MDR Acinetobacter and Enterobacterales in the NICU of a tertiary healthcare center in Surabaya, Indonesia. Methods: This retrospective cohort study was conducted in Dr. Soetomo General Hospital, Surabaya, from January 2020 to August 2022. Documented cases of neonatal sepsis were identified from electronic medical records under the ICD-10 P36 code. Continuous monitoring of blood culture was performed following the initial suspicion of sepsis, complemented with semiautomated antimicrobial susceptibility testing. Results: A total of 75 out of 125 culture-proven neonatal sepsis cases were reported caused by Acinetobacter baumannii ( n = 19; 15.2%) and Enterobacterales ( n = 56; 44.8%) species. These organisms exhibited the MDR rates of 78.9% and 91.1%, respectively. Klebsiella pneumoniae ( K. pneumoniae) emerged as the predominant Enterobacterales, demonstrating an MDR rate of 92.1%. Possible extensively- and possible pan-drug-resistant organisms were also identified at a concerning rate in Acinetobacter (73.7% and 21.1%, respectively) and Enterobacterales (28.6% and 1.8%, respectively). The susceptibility of Acinetobacter to carbapenems was notably low (26.3%), whereas Enterobacterales exhibited a relatively higher susceptibility rate (75%). Notably, 9 and 37 documented deaths were associated with neonatal sepsis because of MDR Acinetobacter and Enterobacterales, respectively. Lower birth weight and shorter length of NICU stay were significantly related to mortality in neonatal sepsis. Conclusion: The alarming rate of MDR underscores further investigations on the extent of inappropriate antibiotic usage in the unit, particularly concerning neonates requiring invasive medical interventions and those with foundational clinical risk factors.