In-use emission rates for a marine vessel operating on hydrotreated algae biofuel are reported for the first time. Emission measurements were made on a four-stroke marine diesel engine from a Stalwart class vessel to compare the emission profile from burning ultra low sulfur diesel (ULSD) to a 50:50 blend of ULSD and algae biofuel (A50). In-use emission measurements followed the International Organization for Standardization (ISO) 8178-4 D2 certification test cycle protocol. Particulate matter (PM2.5), nitrogen oxides (NOx), carbon monoxide (CO), and carbon dioxide (CO2) were sampled in accordance with the ISO 8178-2 protocol for each mode of the test cycle. Switching fuel from ULSD to A50 resulted in significant 35% lower PM2.5 emissions for 25 and 50% load and an overall weighted PM2.5 reduction of 25%. Overall reductions of 30 and 20% were observed in elemental carbon (EC) and organic carbon (OC), respectively. PM2.5 was dominated by OC (77–94%) for both fuels. NOx emissions were reduced by 10% on switching from ULSD to A50. Overall, the emissions of CO2 and CO were reduced by 5 and 18%, indicating a slight improvement in fuel economy for this engine while operating on A50.