Deadly pathogenic multidrug-resistant bacteria (MDR) are becoming more prevalent every day and represent a major danger to human health. This research aimed to isolate and quantify vancomycin resistant MDR (VRMDR) Enterococcus faecalis and extended-spectrum β-lactamase MDR (ESBLMDR) Klebsiella pneumoniae by detecting specific genes and the use of TiO2 Aspergillus oryzae nanoparticles as antibacterial agent against the two strains. One hundred and fifty clinical specimens were collected from Mbarret El-Asafra Hospital, 80 isolates were E. faecalis and 70 isolates were K. pneumoniae. 21/80 was found to be VRMDR E. faecalis, the findings showed that 76.19% of VRMDR E. faecalis strains harboured the VanA gene and 90.47% harboured the VanB gene, while 66.66% of them carried the two resistance genes. On the other hand, 18/70 samples were found to be ESBLMDR K. pneumoniae, the findings showed that 72.22% of ESBLMDR K. pneumoniae strains harboured the blaTEM gene and 61.11% harboured the blaSHV gene, while 33.33% of them carried the two resistance genes. 30µg/ml of nano TiO2 A. oryzae was found to be the minimal inhibitory concentration (MIC) for the VRMDR E. faecalis, while 50µg/ml of nano TiO2 A. oryzae was found to be the MIC for the ESBLMDR K. pneumoniae. The IC50 of TiO2 A. oryzae nanoparticles against human gastric epithelial cell line (GES1) was 563.023±31.7µg/ml compared to chloramphenicol, imipenem drugs and TiO2 nanoparticles showing (563.023±31.7µg/ml, 169.386±9.32µg/ml, 71.692±5.05µg/ml and 30.562±3.22µg/ml) respectively, showing that TiO2 nanoparticles, chloramphenicol and imipenem were more cytotoxic on GES1 normal cells than TiO2 A. oryzae nanoparticles.