Biodiesel presents a promising alternative to conventional diesel fuel, boasting similar combustion characteristics with lower carbon emissions on a well-to-wheel basis. However, the combustion of biodiesel still yields hydrocarbon (HC), carbon monoxide (CO), nitrogen oxides (NOx), and particulate matter (PM) emissions, akin to those from traditional diesel usage. Consequently, aftertreatment systems are imperative to curtail these emissions and comply with increasingly stringent environmental regulations. Diesel oxidation catalysts (DOCs) are pivotal components in modern aftertreatment systems, serving to convert unburned HC and CO, partially convert NO to NO2 to enhance downstream selective catalytic reduction (SCR) catalyst efficiency via fast SCR, and periodically cleanse diesel particulate filters (DPFs) through controlled soot oxidation. This study delves into the performance contrast between biodiesel and diesel over a commercial DOC catalyst to pinpoint knowledge gaps during the transition from diesel to biodiesel fuel. Our findings underscore that the tested biodiesel exhibits a lower light-off temperature compared to diesel. These observations are substantiated by carbon balance analyses and experimental results from HC adsorption and temperature programmed oxidation (TPO) tests. Additionally, the study reveals that the conversion efficiency of certain pollutants, such as HC and CO, is lower with biodiesel than with diesel fuel on the same DOC catalyst. This disparity is attributed to differences in the chemical composition and physical properties of biodiesel, particularly its higher molecular weight and greater oxygen content compared to diesel. In conclusion, elucidating these distinctions between biodiesel and diesel combustion behaviors over DOC catalysts is critical for optimizing aftertreatment system performance and ensuring compliance with evolving emission regulations amidst the transition towards renewable fuel sources.