Background <p>Blood culture contamination is a significant problem that can lead to misdiagnosis, unnecessary antibiotic use, and increased workload. This study aimed to determine the blood culture contamination rates in a tertiary hospital and to evaluate the factors associated with contamination.</p> Methods <p>This retrospective descriptive study included blood culture samples collected at a tertiary hospital between January 2023 and December 2023. Cultures from patients aged ≥ 18 years were evaluated. Growth of coagulase-negative staphylococci (CoNS) and other skin flora agents that were not considered clinically significant were classified as contamination. Contamination risk factors, including the sampling unit, sampling period, and healthcare personnel who collected the cultures, were evaluated.</p> Results <p>A total of 7716 blood cultures from 2388 patients were included. 11.8% of the cultures were defined as contaminated. Contamination rates were found to be significantly higher in internal medicine wards (12.5%) and intensive care units (13.5%) compared to surgical wards (3.6%) (<i>p</i> &lt; 0.001). In logistic regression analysis, the risk of contamination was found to be 3 times higher in blood cultures taken from internal medicine wards and 4.1 times higher in ICUs compared to surgical wards (<i>p</i> &lt; 0.001 and <i>p</i> &lt; 0.001, respectively). Blood cultures obtained by intern doctors (sixth-year medical students) were independently associated with contamination (OR = 1.68; <i>p</i> &lt; 0.001). The risk of contamination was higher in blood cultures taken between July and December (OR = 1.23; <i>p</i> = 0.004).</p> Conclusion <p>Blood culture contamination is a multifactorial problem influenced by factors such as the sampling unit and staff experience. These findings highlight the need for targeted quality improvement interventions, particularly structured training for newly rotating clinical staff.</p> Clinical trial registration <p>Not applicable.</p>