The use of multitracer field fluorometry is increasing in the hydrological sciences. However, obtaining high‐quality fluorescence measurements is challenging given the variability in environmental conditions within stream ecosystems. Here, we conducted a series of stream tracer tests to examine the degree to which multitracer field fluorometry produces reliable estimates of tracer concentrations under realistic field conditions. Using frequently applied examples of conservative (Uranine) and reactive (Resazurin‐Resorufin) fluorescent tracers, we show that in situ measurements of tracer breakthrough curves can deviate markedly from corresponding samples analyzed under laboratory conditions. To investigate the effects of key environmental variables on fluorescence measurements, we characterized the response of field fluorometer measurements to changes in temperature, turbidity, and tracer concentration. Results showed pronounced negative log‐linear effects of temperature on fluorescence measurements for all tracers, with stronger effects observed typically at lower tracer concentrations. We also observed linear effects of turbidity on fluorescence measurements that varied predictably with tracer concentration. Based on our findings, we present methods to correct field fluorometer measurements for variation in these parameters. Our results show how changing environmental conditions can introduce substantial uncertainties in the analysis of fluorescent tracer breakthrough curves, and highlight the importance of accounting for these changes to prevent incorrect inferences being drawn regarding the physical and biogeochemical processes underpinning observed patterns.