Over recent decades, the global occurrence of pesticide residues in aquatic environments has been a pivotal issue; however, their trace-level concentrations necessitate the establishment of ultra-sensitive and reliable analytical approaches. To this end, the present study describes the optimization and validation of an LC-HRMS-based method for the accurate determination of 18 pesticides in river and sea water, accompanied by a measurement uncertainty estimation. This method was applied to analyze 17 real samples from agriculture and aquaculture-impacted areas in Greece and Albania. Different solid-phase extraction (SPE) protocols were tested. For the analysis, cutting-edge Orbitrap MS technology and MS/MS fragmentation, along with the use of matrix-matched calibration curves, provided unprecedented accuracy (<5 ppm) and sensitivity for the confirmation of positive detections. Regarding method performance, exceptional linearity was obtained; the limits of quantification ranged from 1.7 ng L−1 to 90 ng L−1, recoveries varied from 61% to 96% in river water, while slightly higher recoveries (60–111%) were observed in seawater. In all cases, repeatability and intra-laboratory reproducibility were below 15%. The measurement expanded uncertainty (U′, k = 2) was estimated considering precision and bias. MU% values were lower than 50% in all cases, as recommended in SANTE guidelines and applied to the quantified results. The matrix effect study exhibited negative values (<20%) for all compounds. Application to real samples showed a low pesticide contamination load that should not be underestimated.