Clinically relevant antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in bioaerosols have become a greater threat to public health. However, few reports have shown that ARB and ARGs were found in the atmosphere. High-throughput sequencing applied to environmental sciences has enhanced the exploration of microbial populations in atmospheric samples. Thus, five nosocomial bioaerosols were collected, and the dominant microbial and pathogenic microorganisms were identified by high-throughput sequencing in this study. The results suggested that the dominant microorganisms at the genus level were Massilia, Sphingomonas, Methylobacterium, Methylophilus, Micrococcineae, and Corynebacterineae. The most abundant pathogenic microorganisms were Staphylococcus saprophyticus, Corynebacterium minutissimum, Streptococcus pneumoniae, Escherichia coil, Arcobacter butzleri, Aeromonas veronii, Pseudomonas aeruginosa, and Bacillus cereus. The relationship between microbial communities and environmental factors was evaluated with canonical correspondence analysis (CCA). Meanwhile, differences in the pathogenic bacteria between bioaerosols and dust in a typical hospital was investigated. Furthermore, cultivable Staphylococcus isolates with multi-drug resistance phenotype (> 3 antibiotics) in the inpatient departments were much higher than those in the transfusion area and out-patient departments, possibly attributed to the dense usage of antibiotics in inpatient departments. The results of this study might be helpful for scientifically air quality control in hospitals.