Rickettsial diseases are one of the leading causes of treatable acute febrile illness in Asia pacific region. This cross-sectional descriptive study was conducted at Department of Microbiology, Mymensingh Medical College to diagnose scrub typhus by rapid Immunochromatographic Test (ICT) and Nested PCR followed by molecular identification of possible Rickettsial coinfection among suspected febrile patients in Mymensingh, Bangladesh from March 2019 to February 2020. Among the enrolled 402 patients, 89 samples (22.13%) were seropositive by Immunochromatographic Test (ICT) and 65 samples (16.16%) were positive for O. tsutsugamushi DNA by Nested PCR, targeting 47KDa gene. Therefore, 113/402 (28.10%) samples were positive for scrub typhus by PCR and/or ICT. All the scrub typhus positive samples were further subjected to Nested PCR targeting 17 KDa gene for identification of Rickettsial co-infection and 13/113 (11.50%) were documented as positive. Then 13 Rickettsial co-infected samples were undertaken to automate sequencing and all were genetically confirmed as Rickettsia felis. Findings of the study may help clinicians to expand their list of differential diagnoses for undifferentiated fever and detection of Rickettsial co-infection may guide them to prescribe effective antimicrobials.