Abstract Purpose This study aimed to assess the feasibility of the ecoenzymatic stoichiometry and isotope signature approaches as indicators of urban soil functionality, related to carbon and nutrient cycles. Methods In Pisa and Livorno (Italy), study sites with three degrees of urbanization (natural, peri-urban and central urban sites) were selected, where holm oak (Quercus ilex L.) was the most common evergreen species. The urban and peri-urban sites differed in terms of NO2 emissions. At each site, topsoil and plant litter were sampled, pH, EC, TOC, and TN were measured in soil and δ13C and δ15N in soil and plant litter. The β-glucosidase, acid phosphatase and N-acetyl-β-D-glucosaminidase enzyme activities were also determined in soil and the ratios were calculated. Results The δ15N in plant litter increased from peri-urban to urban sites, along with the NO2 emissions, emerging as a sensitive indicator of atmospheric N deposition. The δ15N and δ13C increased in soil, indicating more rapid N cycles and organic matter degradation in peri-urban and urban areas than in natural areas. The ecoenzymatic stoichiometry revealed C and P microbial limitations for all the sites studied. However, the microbial needs of C and P increased and decreased, respectively, along the urbanization gradient. Isotope abundance and microbial nutrient limitations were found to correlate with soil properties. Specifically, soil δ15N was closely correlated with microbial C limitations. Conclusion The isotope signature and enzymatic stoichiometry used as indicators revealed that the soil characteristics affected the soil carbon and nutrient cycles as well as microbial energy and nutrient needs.