Nutrient optimization has been proposed as a way to increase boreal forest production, and involves chronic additions of liquid fertilizer with amounts of micro- and macro-nutrients adjusted annually to match tree nutritional requirements. We used a short-term (maintained since 2007) and a long-term (maintained since 1987) fertilization experiment in northern Sweden, in order to understand nutrient optimization effects on soil microbiota and mesofauna, and to explore the relationships between plant litter and microbial elemental stoichiometry. Soil microbes, soil fauna, and aboveground litter were collected from the control plots, and short- and long-term nutrient optimization plots. Correlation analyses revealed no relationships between microbial biomass and litter nutrient ratios. Litter C:N, C:P and N:P ratios declined in response to both optimization treatments; while only microbial C:P ratios declined in response to long-term nutrient optimization. Further, we found that both short- and long-term optimization treatments decreased total microbial, fungal, and bacterial PLFA biomass and shifted the microbial community structure towards a lower fungi:bacterial ratio. In contrast, abundances of most fungal- and bacterial-feeding soil biota were little affected by the nutrient optimization treatments. However, abundance of hemi-edaphic Collembola declined in response to the long-term nutrient optimization treatment. The relative abundances (%) of fungal-feeding and plant-feeding nematodes, respectively, declined and increased in response to both short-term and long-term treatments; bacterial-feeding nematodes increased relative to fungal feeders. Overall, our results demonstrate that long-term nutrient optimization aiming to increase forest production decreases litter C:N, C:P and N:P ratios, microbial C:P ratios and fungal biomass, whereas higher trophic levels are less affected.