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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) environmental contamination occurs through droplets and biological fluids released in the surroundings from patients or asymptomatic carriers. Surfaces and objects contaminated by saliva or nose secretions represent a risk for indirect transmission of coronavirus disease 2019 (COVID-19). We assayed surfaces from hospital and living spaces to identify the presence of viral RNA and the spread of fomites in the environment. Anthropic contamination by droplets and biological fluids was monitored by detecting the microbiota signature using multiplex quantitative real-time PCR (qPCR) on selected species and massive sequencing on 16S amplicons. A total of 92 samples (flocked swabs) were collected from critical areas during the pandemic, including indoor (three hospitals and three public buildings) and outdoor surfaces exposed to anthropic contamination (handles and handrails, playgrounds). Traces of biological fluids were frequently detected in spaces open to the public and on objects that are touched with the hands (>80%). However, viral RNA was not detected in hospital wards or other indoor and outdoor surfaces either in the air system of a COVID hospital but only in the surroundings of an infected patient, in consistent association with droplet traces and fomites. Handled objects accumulated the highest level of multiple contaminations by saliva, nose secretions, and fecal traces, further supporting the priority role of handwashing in prevention. In conclusion, anthropic contamination by droplets and biological fluids is widespread in spaces open to the public and can be traced by qPCR. Monitoring fomites can support evaluation of indirect transmission risks for coronavirus or other flu-like viruses in the environment. IMPORTANCE Several studies have evaluated the presence of SARS-CoV-2 in the environment. Saliva and nasopharyngeal droplets can land on objects and surfaces, creating fomites. A suitable indicator would allow the detection of droplets or biofluids carrying the virus. Therefore, we searched for viral RNA and droplets and fomites on at risk surfaces. We monitored by qPCR or next generation sequencing (NGS) droplets through their microbiota. Although the study was performed during the pandemic, SARS-CoV-2 was not significantly found on surfaces, with the only exception of environmental areas near infectious patients. Conversely, anthropic contamination was frequent, suggesting a role for biofluids as putative markers of indirect transmission and risk assessment. Moreover, all SARS-CoV-2-contaminated surfaces showed droplets' microbiota. Fomite monitoring by qPCR may have an impact on public health strategies, supporting prevention of indirect transmission similarly to what is done for other communicable diseases (e.g., influenza and influenza-like infections)., (Copyright © 2021 Piana et al.)