The inanimate hospital environment may serve as a reservoir for resistant bacteria that pose nosocomial infection risks often originating from cross contamination and the most common means of pathogens transference occurs between hands of health professionals, hospital equipment and patients. The aim of this study was to investigate various nosocomial infections with multidrug resistant Enterobacteriaceae (MDR-E) and their dissemination in hospital surfaces of two Algerian hospitals from January 2014 to December 2014. Enterobacteriaceae isolated from hospitalized patients and inanimate surfaces were identified by microbiological methods and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOFMS). Antibiotic susceptibility was performed using disk diffusion method. Among 74 nosocomial infections detected, 44 were caused by MDR-E (59%) from different clinical specimen; 23 Klebsiella pneumoniae, 13 Escherichia coli, 5 Enterobacter cloacae and 3 Citrobacter freundii. From inanimate surfaces, MDR-E represents 61% (23 strains out of 38 MDR bacteria isolated); 9 E. cloacae, 8 K. pneumoniae, 4 C. freundii and 2 E. coli. In total, 67 MDR-E were isolated in 2014. Most Enterobacteriaceae show resistance to 13 antibiotics tested out of 15, especially to third-generation cephalosporins, thus resistance to all β-lactams except carbapenems. Here, the dominance of MDR-E isolated from nosocomial infections and in hospital surfaces in Algeria and a characterization of Enterobacteriaceae strains isolated from different specimens according to their species by MALDI-TOF MS were reported. Interestingly, high level of similarity was found between clinical and environmental strains in antibiotic resistance patterns.