Human exposure to BPs is inevitable mostly due to contaminated food. In this preliminary study, for the first time, the presence of bisphenols (BPs) in aqueous humor (AH) collected from 44 patients undergoing cataract surgery was investigated. The measurements were performed using a sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS). Chromatographic separation was achieved using a reverse-phase column and a gradient elution mode. Multiple reaction monitoring (MRM) was used. The method was validated for bisphenol A (BPA) and bisphenol F (BPF). The limits of quantification (LOQs) of both investigated analytes were 0.25 ng mL−1. The method was linear in the range of 0.25–20.0 ng mL−1 with correlation coefficients (R2) higher than 0.98. Recovery of analytes was in the range of 99.9 to 104.3% and intra-assay and inter-assay precision expressed by relative standard deviations (RSD%) were less than 5%. BPA was detected in 12 AH samples with mean concentrations of 1.41 ng mL−1. BPF was not detected at all. Furthermore, two structural isomers termed BPA-1, and BPA-2 were identified, for the first time, in 40.9% of the AH samples, with almost twice higher mean concentrations of 2.15 ng mL−1, and 2.25 ng mL−1, respectively. The total content of BPs were higher in patients with coexisting ocular pathologies such as glaucoma, age-related macular degeneration (AMD), and diabetes in comparison to cataracts alone. However, the difference between these groups did not reach statistical significance (p > 0.05). Performed investigations indicate the need for further research on a larger population with the aim of knowing the consequences of BPs’ accumulation in AH for visual function.