Microorganisms play a significant role in mineral precipitation, but detecting them in the fossil record is still a challenge. Here we offer an example of how the detection of biological activity in the sedimentary environment can modify a classical depositional model. This study describes the activity of microorganisms in sedimentary structures and the iron mineral formation during the last Paleozoic Ice Age in southwestern Gondwana, recorded by the “Itu rhythmites”, Paraná Basin, Brazil. The Itu rhythmites have been considered to be varve-type deposits that present alternating dark laminae (clay/silt-size sediments) and light layers (sand/gravel-size sediments) of varied thickness, forming couplets. Earlier studies focused on abiotic processes of these structures. We applied different techniques and analytical approaches were used, such as synchrotron-based techniques and rock magnetic techniques, in order to test the biogenicity of iron minerals contained in putative microbially-induced sedimentary structures. By detecting biominerals in this rock succession, the depositional model had to be reconsidered, taking into account the biological activity, the limitations on the specific conditions for bacterial growth, and for mineral precipitation. Therefore, we offer a new depositional model that considers the role of microorganisms in formation of these laminae, and this model can be considered for other iron-rich rhythmic deposits in other places of the world. Considering the effects of temperature and other factors in the bacterial productivity, the deposition of the latest couplets in the outcrop occurred in different seasons and by different depositional processes, corroborating with the non-periodicity of 1 year per lithological couplet.