Febrile illnesses contribute significantly to morbidity and mortality in sub-Saharan Africa, but the lack of diagnostic facilities and the broad spectrum of pathogens can lead to inadequate clinical management. The timely and reliable identification of the causative pathogens in febrile patients is the basis for the administration of optimal treatment. We aimed to evaluate the performance of a multiplex polymerase chain reaction (PCR) among blood culture-negative patients presenting with febrile diseases in Central Ethiopia. From April 2016 to June 2018, we collected blood samples from adults and children ≥1 year of age admitted with febrile diseases to the Asella Referral and Teaching Hospital, which is located at an altitude of 2,400 m. Total nucleic acids were extracted from frozen plasma samples using a MagNA Pure 96 instrument (Roche, Mannheim, Germany). The multiplex PCR assays were used in combination with LightCycler multiplex DNA master mix (Roche) on a LightCycler 480 instrument (Roche). We used the pathogen-specific assays targeted to Plasmodium spp., Borrelia spp., Rickettsia spp., Leptospira spp., Salmonella spp., and arboviruses. We tested plasma samples of 511 patients and found positive results for Plasmodium spp. (13, 2.5%), Borrelia spp. (12, 2.3%), and Rickettsia species (7, 1.3%); in total, pathogens were detected in 32 of the samples (6.3%). No pathogen was detected by multiplex PCR in 94% of blood culture-negative samples. Even if the pathogens identified by PCR were not necessarily causes of fever, molecular testing using a multiplex PCR can contribute to pathogen diagnosis in a proportion of febrile patients in the highland part of Ethiopia and help to improve the clinical management.