Proactive interference (PI) occurs when previously learned information impairs memory for more recently learned information. Most PI studies have employed verbal stimuli, while the role of PI in visual working memory (VWM) has had relatively little attention. In the verbal domain, Johansson and colleagues (2018) found that pupil diameter - a real-time neurophysiological index of cognitive effort - reflects the accumulation and resolution of PI. Here we use a novel, naturalistic paradigm to test the behavioral and pupillary correlates of PI resolution for what-was-where item-location bindings in VWM. Importantly, in our paradigm, trials (PI vs. no-PI condition) are mixed in a block, and participants are naïve to the condition until they are tested. This design sidesteps concerns about differences in encoding strategies or generalized effort differences between conditions. Across three experiments (N = 122 total) we assessed PI's effect on VWM and whether PI resolution during memory retrieval is associated with greater cognitive effort (as indexed by the phasic, task-evoked pupil response). We found strong support for PI's detrimental effect on VWM (even with our spatially distributed stimuli), but no consistent link between interference resolution and effort during memory retrieval (this, even though the pupil was a reliable indicator that higher-performing individuals tried harder during memory encoding). We speculate that when explicit strategies are minimized, and PI resolution relies primarily on implicit processing, the effect may not be sufficient to trigger a robust pupillometric response.