Infectious diseases that spread through the bloodstream, known as bloodstream infections (BSIs), are a major global health problem. Positive outcomes for patients with sepsis are typically the result of prompt treatment started after an early diagnosis of BSIs. In this study, we evaluated the capabilities of a portable electronic nose (E-Nose) to detect BSIs with two commonly isolated Gram-negative bacterial species, E. coli and K. pneumonia. One hundred and five blood samples were randomly collected for blood culture examinations using BACTEC and VITEK 2 system, and headspace analysis by an E-Nose from June to December 2021. Classification accuracy of E. coli, K. pneumonia, and negative controls was measured using principal component analysis, area under the receiver operating characteristic curve, sensitivity, and specificity analysis. After incubation for 24 h, cluster plots generated using principal component analysis demonstrated that E-Nose could accurately diagnose the presence of E. coli and K. pneumonia in BACTEC blood culture bottles with a sensitivity and specificity of 100% in just 120 s. The E-Nose method has been shown to be an immediate, precise, and cost-effective alternative to automated blood culture BACTEC and VITEK 2 systems for the fast detection of the causative bacterial pathogens of BSIs in clinical practice. Thus, patients with such Gram-negative bacteremia can have guided empirical antimicrobial therapy on the same day of BSIs diagnosis, which can be lifesaving.