Bacterial infection is a considerable problem in hospitals. Thus, this study was executed to appraise the rampancy of bacterial infections, antimicrobial susceptibility patterns, and molecular characterization of isolates among patients in Bafgh Hospital in Yazd, Iran, in 2020. In the current study, we surveyed 103 isolates of 400 clinical specimens from early March 2020 to September 2020 in Bafgh Hospital. We assessed phenotypic traits and antibiotic resistance with standard microbiological methods. Phenotypic methods were also performed to identify extended-spectrum beta-lactamases (ESBLs) in Gram-negative bacilli, inducible clindamycin resistance, and methicillin resistance in according to CLSI guidelines. Molecular identification of isolates was done by conventional PCR 16S rRNA gene sequencing. Furthermore, we investigated the prevalence of resistant genes including , , , , and in Gram-negative bacteria and the A gene in staphylococcal species. From 400 different clinical specimens, 103 isolates of Gram-positive and Gram-negative bacteria were isolated. Based on phenotypic and molecular methods, most common isolates were (53 isolates), followed by spp. (18 isolates), and (16 isolates). The highest resistance was found in Gram-positive bacteria to erythromycin (66.67%) and penicillin (55.56%), while considering Gram-negative bacteria, the most resistant was cefixime (49.41%) and trimethoprim-sulfamethoxazole (47.05%). In addition, out of 16 isolates, 62.5% and 17.65% were resistant to methicillin and clindamycin, respectively. Among 83 Gram-negative isolates, 22.89% were ESBL-positive. The prevalence of , , , , and genes was 78.31%, 59.03%, 40.96%, 30.12%, and 0%, respectively. The outbreak of bacterial infections is relatively high in hospitals. Recognizing risk agents for bacterial infections and restricting the administration of multidrug-resistant antibiotics is a substantial measure that must be taken to prevent patient mortality.