Currently, biological markers for COVID-19 disease severity still constitute the main goal of enhancing an efficient treatment to reduce critical consequences such as an abnormal systemic inflammatory response. In this regard, the latest research has shown that Pentraxin 3 (PTX3), a highly conserved innate immunity protein, may serve as a valuable biochemical marker. Based on this evidence, we conducted a case-control study to compare the PTX3 serum levels and several immune-inflammatory mediators of 80 healthcare workers who were subdivided into subjects who were previously infected with SARS-CoV-2 ( = 40) and individuals who were never infected ( = 40). Using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA), PTX3 and various immune-inflammatory protein levels were assessed in serum samples, while also considering possible variables (e.g., gender-related differences). We have shown elevated levels of PTX3 and other inflammatory proteins in previously infected COVID-19-positive subjects ( < 0.001). Moreover, the obtained data also indicate a degree of severity influenced by gender, as shown by the subgroup analysis, in which PTX3 expression was more pronounced in previously COVID-19-positive males ( < 0.001) than in females ( < 0.05) compared to the respective controls. In addition, our data further validate, through a direct comparison of previously COVID-19-positive subjects, greater pro-inflammatory levels in males than in females. Overall, our results may support the validity of PTX3 as a systemic biomarker in prolonged systemic inflammatory responses in the context of COVID-19. Thus, PTX3 modulation could constitute an effective therapeutic strategy for improving the recovery from COVID-19 and its systemic long-term consequences.