Tick-borne diseases (TBDs) pose a major hindrance to livestock production in countries with limitedresources. Effective prevention and management of TBDs require a thorough understanding of disease vectorsand pathogens. However, there is limited information on studies of bovine tick-borne pathogens (TBPs) using molecular methods in Malawi. This study aimed to detect TBPs of cattle populations in southern Malawi, which has the largest cattle population in the country. A total of 220 blood samples from apparently healthy cattle were collected in six districts, and werescreened for selected TBPs using polymerase chain reaction (PCR). The overall detection rate of TBPs was 72.3%. Among the detected pathogens, Babesia bigemina hadthe highest detection rate (34.5%), followed by Anaplasma marginale (23.2%), Anaplasma phagocytophilum(22.3%), Theileria taurotragi (22.3%), Theileria parva (15.5%),Anaplasma bovis (9.6%), Babesia bovis (7.3%),Theileria mutans (4.1%), and Babesia naoakii (2.7%). Among the positive samples, 64.2% were found to be co-infected with two or more TBPs, with the highest number of seven pathogens detected in a single sample. Thestudy documents the existence of A. phagocytophilum, B. bovis, and B. naoakii in Malawian cattle for the first time. The findings herein demonstrate a significant burden of TBPs on cattle in Malawi, which gives a challenge in combating TBDs. The high TBP burden, along with the high co-infection frequencies in Malawian cattle necessitates the urgency to implement effective control strategies to enhance cattle production in the country.