The Cottonseed biodiesel combustion, sound and vibrations have been evaluated in a medium duty single cylinder DI engine (1.1L/cyl) by comparison with s ULSD#2 reference values. The engine was supercharged and had 20% EGR and all tests were conducted at 1400 rpm and at 4 bar BMEP load. Cylinder pressure was determined using a Kistler piezoelectric transducer. Combustion pressures peaked at 76 bar for both fuels. Ignition delay for CS100 decreased by 0.16 ms when compared to the ULSD#2 baseline. This would lead to a 23% lower peak heat release rate when operating CS100. The pressure rise rate for CS100 was 20% lower than ULSD#2, which related to the reduced ringing intensity for the biodiesel. The 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, in the whole spectrum from 31Hz to 16 kHz analyzed, reaching a maximum of 3 dBA difference from reference at 82 dBA and 315 Hz one-third octave band and 3.75 dBA at difference from reference 60 dBA in the 100 Hz one-third octave band in the lower frequencies. Cottonseed biodiesel combustion produced higher vibrations in the X and Y axes (crankshaft axis and perpendicular to cylinder and crankshaft axes, respectively) 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 than ULSD and also a quieter injection event while being noisier at intake valve maxim actuation. The RMS of the cycles from noise Campbell plots show a very clear trend of quieter engine during CS100 operation by 1.5dBA compared to ULSD. 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. Higher combustion efficiencies were additionally reflected by lower specific unburned hydrocarbons and carbon monoxide.