Nanometer-scale spheroidal objects (20–200 nm wide) have been widely encountered in geological samples, while their origin remains unclear. First described as “nannobacteria” (Folk 1993), they are more likely associated with various bacterial debris. In order to explore their potential role in degradation and preservation of organic matter (OM) in rocks, this research investigates a range of recent and fossil occurrences of various microbial imprints using electron and atomic force microscopy. Selected samples illustrate different stages in OM cycle and diagenesis, i.e., from production to accumulation before and after lithification. More specifically, effects of oxygen- and light-depleted conditions, which occur naturally at the water–sediment interface, have been evaluated in microbial mats using a specifically tailored laboratory strategy. Recent sediments reveal a close association between a large occurrence of nanometer-scale spheroids and the degree of degradation due to microbial activity. Wide nanometer-scale spheroids appear in fossilized microbial mats and in microbial mats under experimental diagenesis. These samples show that nanometer-scale spheroids are associated with well-preserved OM (e.g., intact bacteria). This corresponds to reduced physicochemical degradation processes. This observation suggests that nanometer-scale spheroids, i.e., in the size range of nannobacteria, occur concomitantly with a significant microbial degradation shown by numerous organic debris (e.g., cell walls). Both recent and fossil samples from anoxic settings show a close association of nanometer-scale spheroids with extracellular polymeric substances (EPS) and bacterial cell walls, and not with algal cell walls or other organic compounds. Because they occur concomitantly with well-preserved organic constituents, nanometer-scale spheroids indicate a reduced level of OM degradation in the diagenetic processes. Therefore, nanometer-scale spheroids would be a microbial evidence of early diagenesis in sedimentary rocks.