Clinical or microbiological factors potentially associated with prolonged COVID-19 PCR positivity are still poorly underexplored, but they could be of importance for public-health and clinical reasons. The objective of our analysis is to explore demographic, clinical, and microbiological factors potentially associated with a prolonged positivity to SARS-CoV-2 among 222 hospitalized patients. Prolonged detection positivity for SARS-CoV-2 RNA in swap samples, defined as positivity more than 21 days, was the outcome of interest. The 56 cases with a prolonged positivity to SARS-CoV-2 were matched for age and sex with 156 controls. The cases reported a significantly higher presence of diabetes mellitus, autoimmune diseases, chronic kidney diseases, and acute coronary syndrome. Moreover, the viral load was significantly higher in a period of prolonged positivity compared to a normal period. In the multivariable analysis, the presence of autoimmune diseases and chronic kidney disease were significantly associated with an increased risk of prolonged positivity as well as medium viral load or high viral load, i.e., low Ct value ≤ 30 indicating high viral load. The results of this study confirmed that in a large population of hospitalized patients with COVID-19 manifestations, the prolonged positivity of SARS-CoV-2 detection with nasopharyngeal swab was mainly related to autoimmune diseases, chronic kidney disease, and to baseline viral load.