Wild and cultivated olives in the Western Cape of South Africa are direct or indirect hosts to a high diversity of Braconidae and Chalcidoidea wasps. Olive-associated Braconidae are known to parasitise the larvae of Bactrocera oleae (Rossi), and probably also Bactrocera biguttula (Bezzi). The lifestyle of olive-associated Chalcidoidea is not fully understood, and may include phytophagous, parasitoid and hyperparasitoid species. Some chalcids could represent olive seed wasps (OSW), a generic term that designates the seed feeders responsible for losses in commercial olive production. In order to obtain direct DNA-based evidence for the lifestyle of four putative OSW – Eupelmus spermophilus Silvestri, Eurytoma oleae Silvestri, Eurytoma varicolor Silvestri, Sycophila aethiopica (Silvestri); and the parasitoid Neochrysocharis formosus (Westwood) – we developed a multiplex PCR assay comprising species-specific primers for each species. The assay was used to survey larvae collected from the seeds of wild and cultivated olives. Eupelmus spermophilus was the most abundant species in wild and cultivated olives, and may represent a threat to the local olive industry. Eurytoma oleae was found only within the seeds of wild olives, and may also represent an OSW. We found direct evidence supporting the potential agricultural relevance of S. aethiopica and E. varicolor as parasitoids of E. spermophilus. These results may inform strategies for the management and control of OSW in the Western Cape.