Quantifying the pathways of fixed nitrogen (N) loss in marine oxygen deficient zones (ODZs) and the isotopic fractionation caused by these processes are important for understanding the marine fixed N budget and its potential for change. In this study, a variety of approaches were used to quantify fixed N loss in the eastern tropical South Pacific Ocean (ETSP). The required measurements included nutrient concentration (nitrate—NO3-, nitrite—NO2-, and phosphate—PO43-), gas ratio (N2/Ar) measurements, and stable N and O isotopes in NO3-, NO2-, and nitrogen gas (N2). The dissolved inorganic nitrogen deficit calculated from [PO43-] ([DIN]def,P) exceeded the concentration of N2 gas biologically produced in the ODZ (local [N2]bio) throughout the ODZ at most stations, likely due to release of PO43- from sediments driving up [DIN]def,P. Calculating DIN deficit using water mass analysis and local oxygen (O2) consumption ([DIN]def,OMP) yielded better agreement with local [N2]bio than [DIN]def,P, except at the maximum [N2]bio, where [DIN]def,OMP misses contributions of anaerobic ammonia oxidation (anammox) to N2 production. We used the mismatch between [DIN]def,OMP and [N2]bio to estimate a 29% contribution of anammox to [N2]bio. Stable isotopic measurements of NO2-, NO3-, and N2 were used alongside [N2]bio and new estimates of [DIN]def to calculate N and O isotope effects for NO3- reduction (15εNAR and 18εNAR, respectively), and N isotope effects for DIN removal (15εDIN-R). While the various methods for estimating [DIN]def had little effect on the isotope effects for DIN removal, differences between 15εNAR and 15εDIN-R, and variations with depth in the ODZ were observed. Using a simple time-dependent ODZ model, we interpreted these patterns to reflect the influences of NO2- oxidation and NO2- accumulation on expression of isotopic fractionation in the ODZ.