Somatic embryogenesis (SE) is the process by which embryo-like structures are formed from somatic cells. Although SE has been successfully applied to several herbaceous annual species, most woody species, including olive (Olea europaea L.), remain recalcitrant, exhibiting low capacity for somatic embryo development, particularly when adult tissues are used as initial explants. To overcome these limitations, a deeper understanding of the molecular mechanisms underlying SE is essential. In olive, however, these mechanisms remain largely unexplored, and the pathways controlling induction of SE and further embryonic differentiation and development are still poorly understood. To contribute to this knowledge, high- and low-embryogenic efficiency lines were established using SE cultures of the olive cultivar ‘Arbequina’, with zygotic embryos used as initial explants. Somatic embryogenic lines were maintained in Embryogenesis Cyclic Olive (ECO). At the end of the third subculture, plant material was collected, and whole proteome analysis was performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), applying both Suspension-Trapping (S-Trap)- and Orbitrap Mass Spectrometer (OT-2)-based workflows. A total of 13,583 proteins and 9,119 protein groups were identified using the S-Trap method, while 13,686 proteins and 9,211 protein groups were identified using the OT-2 method. From these, several well-known SE biomarkers were identified. Isoforms from Class III peroxidases were identified as characteristic of high-embryogenic efficiency lines. These findings provide novel molecular insights into the regulation of SE in olive and may support the development of improved propagation protocols for recalcitrant genotypes.