Healthcare-related infections (HAI) represent a great risk to their victims and are an increasing cost to the clinical and therapeutic setting. The fast increase in microbial resistance to antibiotics in the hospital environment decreases drug effectiveness, increases length of stay, and increases the cost of treatment. Besides that, it may result in premature death as a direct consequence of unsafe health practices, according to the World Health Organization. In this context, the present study aimed to evaluate microbial resistance and biofilm formation in isolates of clinical interest. The methodology used was based on the analysis and identification of clinical isolates of Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis and Providencia stuartii, performed by the Vitek® 2 Compact automated system (bioMérieux) and confirmed by obtaining mass spectrum in MALDI-TOF apparatus. Autoflex III Mass Spectrometer which were compared with MALDI Biotyper version 3.1 database. Microbial resistance was evaluated by Minimum Inhibitory Concentration (MIC) and biofilm formation of these isolates by violet crystal method, Congo Red Agar and optical microscopy. As results, it was observed that all isolates tested were compatible with identification by MAL-DI-TOF. In addition, of all isolates tested, 83.3% had higher resistance to the antibiotic Ampicillin (AMP) and 58.3% showed resistance to Ciprofloxacin (CIP), ranging from Resistant Multidrug (MDR) to Extensively Drug Resistant (XDR), by various other antibiotics. All microorganisms presented biofilm formation ranging from moderate to strong, in which the strong ones represented 66.6% of isolates. It was observed that the identification of pertinent characteristics to microorganism might provide an effective therapy focused in reducing the infectious diseases related to HAI.