Background: Delays in appropriate antimicrobial therapy can increase the risk of all-cause mortality (ACM) in hospital-acquired bloodstream infections (HA-BSIs) caused by Gram-negative bacteria (GNB). We aimed to evaluate the effectiveness of early appropriate antimicrobial therapy (EAAT) compared with late appropriate antimicrobial therapy (LAAT) in this population. Methods: This study used data from a multi-centre, prospective cohort including adult patients with HA-BSI caused by GNB across 22 Turkish hospitals. A hypothetical target trial allocating participants with HA-BSI caused by monobacterial GNB to either EAAT or LAAT was emulated using the weighting approach. The primary outcome was 14 day ACM; 28 day ACM was a secondary outcome. Cox proportional hazards models with inverse probability weighting were applied to account for confounding, with antibiotic treatment incorporated as a time-dependent variable. Results: Among 680 patients, 14 day ACM occurred in 14.7% (40/272) of the EAAT group and 42.9% (175/408) of the LAAT group. EAAT was associated with a lower risk of 14 day ACM [adjusted hazard ratio (aHR) = 0.41; 95% CI: 0.28–0.61). Similarly, 26.1% (71/272) of the patients treated with EAAT and 49.5% (202/408) of those receiving LAAT died during 28 day follow-up (aHR = 0.66; 95% CI: 0.50–0.86). In the carbapenem-resistant GNB subset, EAAT reduced the hazard of 14 day ACM (aHR = 0.36; 95% CI: 0.21–0.60) and 28 day ACM (aHR = 0.60; 95% CI: 0.44–0.84). All prespecified and post hoc analyses consistently supported these findings. Conclusions: EAAT was associated with a survival benefit in individuals with HA-BSI due to GNB. Although these findings support early initiation of appropriate therapy, residual confounding cannot be excluded.