In this study, gaseous emissions, sound, and vibration levels were analyzed for a single-cylinder, medium duty, DI research diesel engine fueled with 100% Cottonseed biodiesel (CS100) with reference to ULSD#2. The engine was supercharged and had 20% EGR. All tests were conducted at 1400 rpm and at 4 bar IMEP load. CS100 combustion produced a decrease of 37% in NOx and 7.5% in soot compared with ULSD baseline. Sound and vibrations were measured using a B&K condenser type multi-field microphone, and a tri-axial, piezoelectric accelerometer. All noise & vibration signals were analyzed with CPB and FFT Analysis and Crank Angle Domain Analysis with B&K Pulse Platform software. In the CPB analysis, ULSD#2 produced a higher sound level than Cottonseed biodiesel, analyzed in the whole spectrum from 31Hz to 16kHz, reaching a maximum difference of 3.75 dBA at 100 Hz. Higher radial vibrations were observed for CS100 at the peak of 23 Hz, which is the fundamental frequency of the engine. Sound Campbell plots over the duration of the cycle showed that CS100 has a quieter combustion and lower noise from piston slap and also a quieter injection event while being noisier at intake valve maximum actuation. The RMS of the cycles from noise Campbell plots show a clear trend of quieter engine combustion during CS100 operation by 1.5dBA. On the vibrations side, the RMS of Campbell plots show lower vibrations by 1g for CS100. Overall results suggest that the Cottonseed biodiesel produces lower overall sound and vibrations during the combustion process compared to ULSD#2 while decreasing both NOX and soot.