The oldest known evidence of large impacts in Early Earth history are Archaean to Palaeoproterozoic impact spherule layers of the Barberton Greenstone Belt in South Africa and the Pilbara craton in Western Australia. Four drill core sections (BH5901, BH5907, BH5911 and BH5949) containing spherule layers were intersected in exploration drilling at Fairview Gold Mine in the northern Barberton Greenstone Belt. Here, we provide a first detailed mineralogical study of these new spherule layer intersections. The spherule layers are densely packed with sand-sized, spherical to ovoid, i.e., in part strongly deformed, “beads”. They generally have morphological and textural features similar to other 3.4–3.2 Ga old spherule layers of the Barberton Greenstone Belt. The Fairview spherule layers are stratigraphically positioned at the contact between the silicified volcanic tuff of the uppermost unit of the Onverwacht Group and the lowermost siliciclastic rocks of the Fig Tree Group, like the S2 and S4 layers in the southern, and S3 layer in the northern, domain of the greenstone belt. Micro-XRF scanning allowed (i) to distinguish spherule layers from their host rocks by their major element distribution, and (ii) to characterize secondary alteration and micro-deformation features in the SL and host rocks. Ni–Cr spinel, the only primary mineral in evidence in these sections, is not distributed uniformly along a spherule layer intersection, and occurrences can not be stratigraphically correlated between the four layer intersections either. Spinel crystals vary with respect to Ni, Cr, Fe and Zn contents and respective zonation patterns for these elements. Zinc abundances are likely related to post-depositional hydrothermal overprint. The results of this investigation comprising geological, petrographic, semiquantitative micro-X-ray fluorescence spectrometry, and quantitative electron microprobe analysis on spinel support a complex and heterogeneous genetic process that must have taken place during spherule formation in an impact vapor plume and in post-depositional times. The Fairview spherule layers are thought to possibly represent the product of a single impact event. They represent fallout into deep-water conditions, as they lack evidence for reworking or sorting by current or wave action. The heterogeneous Ni–Cr spinel concentrations are similar to what has been reported for spinel occurrences in the S3 layer, but considering the complex tectonic overprint on this lithostratigraphic sequence at the changeover from the Onverwacht to the Fig Tree Group it is not possible to conclude whether or not these spherule layers represent the same impact event.