This paper presents a new version of the 3D mesa Bridge microdosimeter comprised of an array of 4248 silicon cells fabricated on 10 μm thick n-type silicon-on-insulator substrate. This microdosimeter has been designed to overcome limitations existing in previous generation silicon microdosimeters and it provides well-defined sensitive volumes and high spatial resolution. The charge collection characteristics of the new 3D mesa microdosimeter were investigated using the ANSTO heavy ion microprobe, utilizing 5.5 MeV He2 + ions. Measurement of microdosimetric quantities allowed for the determination of the relative biological effectiveness of 290 MeV/u and 350 MeV/u 12C heavy ion therapy beams at the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan. The microdosimetric RBE obtained showed good agreement with the tissue-equivalent proportional counter. Utilizing the high spatial resolution of the SOI microdosimeter, the LET spectra for 70 MeV 12C+6 ions, like those present at the distal edge of 290 and 350 MeV/u beams, were obtained as the ions passed through thin layers of polyethylene film. This microdosimeter can provide useful information about the lineal energy transfer (LET) spectra downstream of the protective layers used for shielding of electronic devices for single event upset prediction.