Stable isotope natural abundance measurements integrate across several biogeochemical processes in ecosystem N and C dynamics. Here, we report trends in natural isotope abundance (δ13C and δ15N in plant and soil) along a climosequence of 33 Nothofagus forest stands located within Patagonia, Southern Argentina. We measured 28 different abiotic variables (both climatic variables and soil properties) to characterize environmental conditions at each of the 33 sites. Foliar δ13C values ranged from −35.4‰ to −27.7‰, and correlated positively with foliar δ15N values, ranging from −3.7‰ to 5.2‰. Soil δ13C and δ15N values reflected the isotopic trends of the foliar tissues and ranged from −29.8‰ to −25.3‰, and −4.8‰ to 6.4‰, respectively, with no significant differences between Nothofagus species (Nothofagus pumilio, Nothofagus antarctica, Nothofagus betuloides). Principal component analysis and multiple regressions suggested that mainly water availability variables (mean annual precipitation), but not soil properties, explained between 42% and 79% of the variations in foliar and soil δ13C and δ15N natural abundance, which declined with increased moisture supply. We conclude that a decline in water use efficiency at wetter sites promotes both the depletion of heavy C and N isotopes in soil and plant biomass. Soil δ13C values were higher than those of the plant tissues and this difference increased as annual precipitation increased. No such differences were apparent when δ15N values in soil and plant were compared, which indicates that climatic differences contributed more to the overall C balance than to the overall N balance in these forest ecosystems.