New Delhi Metallo-β-lactamase (NDM) belongs to the B1 super family of metallo-β-lactamase and is rapidly disseminated globally. NDM producing pathogens confer resistance against a wide range of antibiotics including carbapenems; considered to be the last resort to treat such pathogens. Currently, therapeutic treatment of infections by these bacteria is becoming a big challenge. Manuka honey is the one that is widely used medicinal honey for wound dressing worldwide. Several factors are responsible for their antibacterial activity including high osmolality, pH, hydrogen peroxide concentration, phytochemical nature and methylglyoxal. Therefore, we planned to determine the molecular characterization of NDM producing bacteria and in vitro and in vivo efficacy of Manuka honey against NDM positive strain. A total of 302 carbapenem resistant Gram negative bacteria were collected from four different tertiary care hospitals Lahore. Isolates were reconfirmed by Vitek 2 system and MALDI-TOF. Carbapenamases and MBLs detection was done as per CLSI guidelines 2015. Bacteria were further analyzed for the presence of blaNDM by PCR and NDM variants were detected after Sanger sequencing. MIC (μg/mL) of the NDM positive bacteria were done by Vitek 2 system. MLST of NDM positive K. pneumoniae and E. coli was done by amplifying and sequence their house keeping genes. Plasmids characterization of NDM producing bacteria was done by S1 nuclease PFGE and In Gel DNA-DNA hybridization. In vitro activity of Manuka honey was done by screening the NDM positive bacteria by agar well diffusion method. MIC and MBC (%v/v) of Manuka honey against NDM positive pathogens was done by microbroth dilution method. In vivo activity of Manuka honey was done using BALB/c mouse model by intravenous injection of 20% and 30% of Manuka honey against NDM-1 positive K. pneumoniae strain. Bacterial load (CFU) was counted using Miles and Misra method after seven and fifteen days of clinical trial. Out of 302 clinical isolates, K. pneumoniae (29.8%) were identified followed by A. baumannii (28%) and E. coli (11%) in four tertiary care hospitals. Overall, highest prevalence (38.7%) of carbapenem resistant bacteria was observed in the Children hospital and culprit pathogen were K. pneumoniae (31.6%). Generally, 26.5% of the carbapenem resistant bacteria were recovered from blood culture, 25.4% from urine culture and 22.8% from pus swabs. Among these pathogens, 91% were carbapenamase and 89% were MBLs producers. After molecular identification, 144 (47.6%) bacteria were positive for NDM with majority of K. pneumoniae (52%) and E. coli (15.2%). Multi-sequence alignment revealed four different variants of NDM (NDM-1, 4, 5, and 7) and mainly these variants were found in K. pneumoniae and E. coli with the majority of NDM-1 (82.6%). Overall 100% of the NDM positive bacteria showed highest MIC (μg/mL) against commonly used antibiotics including meropenem, 70%-80% to moxifloxacin, aztreonam, cefepime and levofloxacin while most effective drugs were colistin and tigecycline. Most of the K. pneumoniae belonged to the sequence type (ST) of ST11, ST147 and ST29 however majority of the E. coli belonged to ST405 and ST101. Plasmids characterization displayed most of the isolates (n=130; 90%) carried blaNDM on their plasmids and 6 (4%) carried on chromosome however about 17% of the blaNDM carried on ~150kb of plasmids. Overall, in vitro activity of Manuka honey against 144 isolates exhibited that 55 (38%) NDM positive bacteria were inhibited at the concentration of 30% (v/v), 52 (36%) at 25% (v/v) and 30 (20.8%) at 20% (v/v). Highest MIC was observed against K. pneumoniae (35% v/v) and least observed to C. freundii and M. morganii (15% v/v). In vivo activity of Manuka honey against K. pneumoniae ST11 showed that there was a substantial reduction in the mean log value of each honey treated group as compared to the positive control group (Group B). The mean log value for Group C and D significantly and gradually reduced from 1.829 to 1.035 and 1.624 to 0.610 in first and second week respectively. Statically no significant difference was observed between the treatment Group C and D. This study concluded that there is an alarming spread of blaNDM and its variants in the Gram-negative bacteria particularly among children that displayed resistance to most of the drugs including carbapenems which can lead to treatment failure and mortality. In vitro Manuka honey has the potential effect against the NDM producing pathogens however in vivo the therapeutic effect of Manuka honey gradually and effectively reduces the bacterial load of K. pneumoniae ST11. Therefore, Manuka honey could be used as one of the remedy to treat such pathogens following several in vitro and in vivo clinical studies. Hence, it is need of the hour to conduct a national multicenter survey to combat this serious threat in our hospitals and also extensively look to treat these pathogens with Manuka honey.