Extraordinarily well-preserved pithonellid microfossils (calcitarchs, “calcispheres”) from the Turonian (upper Cretaceous) of Tanzania reveal previously unknown morphological traits, crystallographic patterns, and chemical signatures, providing new insight to this enigmatic group of microfossils. Using combined transmitted-reflected light microscopy, scanning electron microscope imagery, electron microprobe elemental analysis and stable isotope geochemistry, the present study reveals four new aspects of the genus Pithonella, notably, the following. An affinity with cyst-forming organisms, potentially the dinoflagellates, is indicated by the presence of a hatch opening and corresponding operculum. The pristine outer wall architecture consists of thin, smooth shingle-shaped plates with regular rows of slit-shaped pores and an apical sub-angular or circular pore. This primary surface pattern is significantly different from previous descriptions of an outer wall consisting of “parquet-shaped” prismatic crystal rows; this latter surface pattern is formed by secondary overgrowth. The crystallographic pattern of the inner wall is crypto-crystalline. Unaltered pithonellids reveal a calcite chemistry characterized by comparably high Mg-contents, relatively enriched stable carbon isotope values, and stable oxygen values indicating a surface water habitat. Based on these previously unseen traits, the diagnosis of the genus Pithonella is emended. A new species, Pithonella diconica, is described from the lower-middle Turonian sediments of Tanzania.