Antimicrobial Resistance (AMR) caused by Carbapenem-Resistant Enterobacteriaceae (CRE) is a global threat. Accurate identification of these bacterial species with associated AMR is critical for their management. While highly accurate methods to detect CRE are available, they are costly, timely and require expert skills, making their application infeasible in low-resource settings. Here, we investigated the potential of Near-Infrared Spectroscopy (NIRS) for a range of applications: (i) the detection and differentiation of isolates of two pathogenic Enterobacteriaceae species, Klebsiella pneumoniae and Escherichia coli, and (ii) the differentiation of carbapenem resistant and susceptible K. pneumoniae. NIRS has successfully differentiated between K. pneumoniae and E. coli isolates with a predictive accuracy of 89.04% (95% CI; 88.7–89.4%). K. pneumoniae isolates harbouring carbapenem-resistance determinants were differentiated from susceptible K. pneumoniae strains with an accuracy of 85% (95% CI; 84.2–86.1%). To our knowledge, this is the largest proof of concept demonstration for the utility and feasibility of NIRS to rapidly differentiate between K. pneumoniae and E. coli as well as carbapenem-resistant K. pneumoniae from susceptible strains.