Abstract For data assimilation to provide faithful state estimates for dynamical models, specifications of observation uncertainty need to be as accurate as possible. Innovation-based methods based on Desroziers diagnostics, are commonly used to estimate observation uncertainty, but such methods can depend greatly on the prescribed background uncertainty. For ensemble data assimilation, this uncertainty comes from statistics calculated from ensemble forecasts, which require inflation and localization to address under sampling. In this work, we use an ensemble Kalman filter (EnKF) with a low-dimensional Lorenz model to investigate the interplay between the Desroziers method and inflation. Two inflation techniques are used for this purpose: 1) a rigorously tuned fixed multiplicative scheme and 2) an adaptive state-space scheme. We document how inaccuracies in observation uncertainty affect errors in EnKF posteriors and study the combined impacts of misspecified initial observation uncertainty, sampling error, and model error on Desroziers estimates. We find that whether observation uncertainty is over- or underestimated greatly affects the stability of data assimilation and the accuracy of Desroziers estimates and that preference should be given to initial overestimates. Inline estimates of Desroziers tend to remove the dependence between ensemble spread–skill and the initially prescribed observation error. In addition, we find that the inclusion of model error introduces spurious correlations in observation uncertainty estimates. Further, we note that the adaptive inflation scheme is less robust than fixed inflation at mitigating multiple sources of error. Last, sampling error strongly exacerbates existing sources of error and greatly degrades EnKF estimates, which translates into biased Desroziers estimates of observation error covariance. Significance Statement To generate accurate predictions of various components of the Earth system, numerical models require an accurate specification of state variables at our current time. This step adopts a probabilistic consideration of our current state estimate versus information provided from environmental measurements of the true state. Various strategies exist for estimating uncertainty in observations within this framework, but are sensitive to a host of assumptions, which are investigated in this study.