Acinetobacter species have emerged as one of the leading causative agents of Hospital-Acquired Infections (HAIs). Acinetobacter baumannii, the most commonly isolated clinical subspecies among the Acinetobacter species, is known to cause a wide range of HAIs, with an increased risk of mortality ranging from 8% to 40% in hospitalized patients. This study aimed to evaluate the clinical significance of Acinetobacter species isolated from admitted patients, ward environmental surfaces and instruments at the University Teaching Hospital, (UTH) in Lusaka, Zambia.This was a facility-based cross-sectional study that involved collecting clinical samples (cerebrospinal fluid, sputum, pus swabs, blood culture, urine, synovial fluid, pericardial fluid, and pleural fluid) that were submitted to the Microbiology Laboratory at UTH as part of the routine diagnosis and patient care between July and December 2021. Environmental specimens were also collected from surfaces and equipment in contact with patients, such as beds, beddings, suction machines, ventilators, ward gowns and endotracheal tubes.Sixty Acinetobacter isolates were recovered, 40 (66.7%) from clinical specimens and 20 (33.3%) from environmental samples, respectively. Among the clinical isolates, the admission ward had the highest (21.7%; 95% CI: 10.9–32.4) while the main ICU had the least (1.7%; 95% CI: 0.0–5.0%); for environmental samples, the highest isolation was from taps (25%) and sinks (20%). Among the sixty Acinetobacter isolates, A. baumannii was the most prevalent subspecies (n = 43). Highest resistance was observed to tetracycline (98%; 95% CI: 94–100) and co-trimoxazole (70%; 95% CI: 58–82) while the lowest resistance was seen in imipenem (17%; 95% CI: 7.3–28.3), tobramycin (20%; 95%CI: 11.7–36) and cefotaxime (22%; 95% CI: 24–22). The AdeB gene (efflux pump) was detected in 82.5% of the clinical isolates. The frequency of clinical isolates that showed resistance to at least three classes of antibiotics (aminoglycosides, fluoroquinolones and sulphonamides) translated into being Multidrug-resistant (MDR) and Extensive drug resistance (XDR) in the clinical isolates was 75% (30/40) and 52.5% (21/30), respectively. These findings intimate the clinical significance of HAI Acinetobacter infections and need for continuous AMR surveillance to inform treatment guidelines and regular infection prevention processes.