Diamond is a promising platform for the development of technological applications in quantum optics and photonics. The quest for color centers with optimal photo-physical properties has led in recent years to the search for novel impurity-related defects in this material. Here, we report on a systematic investigation of the photo-physical properties of two He-related (HR) emission lines at 535.2 nm and 559.7 nm (as measured at a temperature of 25 K) created in three different diamond substrates upon implantation with 1.3 MeV He+ ions and subsequent annealing. The spectral features of the HR centers were studied in an “optical grade” diamond substrate as a function of several physical parameters, namely, the measurement temperature, the excitation wavelength, and the intensity of external electric fields. The emission lifetimes of the 535.2 nm and 559.7 nm lines were also measured by means of time-gated photoluminescence measurements. The Stark shifting of the HR centers under the application of an external electrical field was observed in a CVD diamond film equipped with buried graphitic electrodes, suggesting the lack of inversion symmetry in the defects' structure. Furthermore, the photoluminescence mapping under 405 nm excitation of a “detector grade” diamond sample implanted at a He+ ion fluence of 1 × 1010 cm−2 enabled us to identify the spectral features of both the HR emission lines from the same localized optical spots. The reported results provide an insight into the structure of He-related defects in diamond and their possible utilization in practical applications.