The hospital environment significantly contributes to the onset of healthcare associated infections (HAIs), representing the most frequent and severe complications related to health care. The monitoring of hospital surfaces is generally addressed by microbial cultural isolation, with some performance limitations. Hence there is need to implement environmental surveillance systems using more effective methods. This study aimed to evaluate next-generation sequencing (NGS) technologies for hospital environment microbiome characterization, in comparison with conventional and molecular methods, in an Italian pediatric hospital. Environmental samples included critical surfaces of randomized rooms, surgical rooms, intensive care units and delivery rooms. The resistome of the contaminating population was also evaluated. NGS, compared to other methods, detected with higher sensitivity the environmental bacteria, and was the only method able to detect even unsearched bacteria. By contrast, however, it did not detect mycetes, nor it could distinguish viable from dead bacteria. Microbiological and PCR methods could identify and quantify mycetes, in addition to bacteria, and PCR could define the population resistome. These data suggest that NGS could be an effective method for hospital environment monitoring, especially if flanked by PCR for species identification and resistome characterization, providing a potential tool for the control of HAI transmission.