Rodents serve as reservoirs and/or vectors for several human infections of high morbidity and mortality in the tropics. Population growth and demographic shifts over the years have increased contact with these mammals, thereby increasing opportunities for disease transmission. In Africa, the burden of rodent-borne diseases is not well described. To investigate human seroprevalence of selected rodent-borne pathogens, sera from 657 healthy adults in ten rural communities in Ghana were analyzed. An in-house enzyme-linked immunosorbent assay (ELISA), for immunoglobulin G (IgG) antibodies to Lassa virus was positive in 34 (5%) of the human samples. Using commercial kits, antibodies to hantavirus serotypes, Puumala and Dobrava, and Leptospira bacteria were detected in 11%, 12% and 21% of the human samples, respectively. Forty percent of residents in rural farming communities in Ghana have measurable antibodies to at least one of the rodent-borne pathogens tested, including antibodies to viral hemorrhagic fever viruses. The high seroprevalence found in rural Ghana to rodent-borne pathogens associated with both sporadic cases and larger disease outbreaks will help define disease threats and inform public health policy to reduce disease burden in underserved populations and deter larger outbreaks.