Microbial communities in human-impacted soils of ancient settlements have been proposed to be used as ecofacts (bioindicators) of different ancient anthropogenic activities. In this study, bacterial, archaeal and fungal communities inhabiting soil of three archaic layers, excavated at the archaeological site on Monte Iato (Sicily, Italy) and believed to have been created in a chronological order in archaic times in the context of periodic cultic feasts, were investigated in terms of (i) abundance (phospholipid fatty acid (PLFA) analysis and quantitative PCR)), (ii) carbon(C)-source consumption patterns (Biolog-Ecoplates) and (iii) diversity and community composition (Illumina amplicon sequencing). PLFA analyses demonstrated the existence of living bacteria and fungi in the soil samples of all three layers. The upper layer showed increased levels of organic C, which were not concomitant with an increment in the microbial abundance. In taxonomic terms, the results indicated that bacterial, archaeal and fungal communities were highly diverse, although differences in richness or diversity among the three layers were not detected for any of the communities. However, significantly different microbial C-source utilization patterns and structures of bacterial, archaeal and fungal communities in the three layers confirmed that changing features of soil microbial communities reflect different past human activities.