Background: Central line-associated bloodstream infections (CLABSIs) are a significant healthcare challenge globally, increasing mortality risk and complicating central vascular catheter use. In Colombia, few studies have assessed the impact of CLABSIs on hospital stay and mortality. Objective: To determine the association between CLABSIs and discharge outcomes and hospital stay duration in adult patients at a tertiary care institution in Cali, Colombia, from 1 January 2015 to 31 December 2018. Methods: A nested case-control study was conducted. The odds of mortality associated with CLABSIs were estimated using conditional logistic regression. Non-conditional logistic regression was used to determine the odds of mortality when CLABSIs were caused by resistant microorganisms. Hospital stay duration, catheter duration, and time from catheter insertion to discharge were compared between patients with and without CLABSIs. The most frequent etiological agents were identified. Results: Patients with CLABSIs had 3.89 times the odds of mortality (95% CI [1.33-11.31], p = 0.013) compared to those without CLABSIs. The odds of mortality for patients with resistant microorganism CLABSIs were 4.04 times (95% CI [1.17-13.96], p = 0.027) higher than those with sensitive microorganism CLABSIs. Hospital stay duration (median = 51 days vs. 17 days; p = 0.000), catheter duration (median = 19 days vs. 7 days; p < 0.001), and time from catheter insertion to discharge (median = 40 days vs. 9 days; p < 0.001) were significantly longer in CLABSI patients. Klebsiella pneumoniae was the most isolated pathogen (20.2%), followed by Staphylococcus aureus (14.9%). Implications: CLABSI patients have longer catheter and hospitalization durations and higher mortality risk. Resistant microorganism CLABSIs are associated with elevated mortality risk. Conclusions: This study corroborates the positive relation between CLABSI and the mortality risk, which is influenced by resistant bacteria, though causality is not established. CLABSI is also linked to longer hospital stays, underscoring the need for improving infection control strategies.