Pneumocystis jirovecii (P. jirovecii) is an opportunistic fungus responsible for Pneumocystis pneumonia (PCP) in deeply immunocompromised patients and for pulmonary colonization in individuals with mild immunosuppression or impaired respiratory function. PCP and Cytomegalovirus (CMV) co-infections have been widely described whereas those involving other Herpesviruses (HVs) such as Epstein-Barr virus (EBV), Herpes simplex virus type 1 and type 2 (HSV-1 and  -2), and Varicella zoster virus (VZV) remain scarce. To date, no data are available concerning HVs co-infections in P. jirovecii colonization. Our main objective was to evaluate the frequency of HVs in bronchoalveolar lavage fluid (BALF) samples from patients with PCP or with pulmonary colonization. The secondary objective was to assess the relationship between HVs and the mortality rate in PCP patients. A retrospective single-center study over a seven-year period was conducted. All patients with P. jirovecii detected using PCR in a BALF sample and for whom a PCR assay for HVs detection was performed were included in the study. One hundred and twenty-five patients were included, corresponding to 77 patients with PCP and 48 colonized patients. At least one HV was detected in 54/77 (70.1%) PCP patients and in 28/48 (58.3%) colonized patients. EBV was the most frequent in both groups. Furthermore, the 30-day survival rate in PCP patients was significantly lower with EBV + CMV co-infection than that with EBV co-infection, EBV + HSV-1 co-infection and without HV co-infection. Our results show that the frequency of HV, alone or in combination is similar in PCP and colonization. They also suggest that EBV + CMV detection in BALF samples from PCP patients is associated with an increased mortality rate, underlying the significance to detect HVs in the course of PCP.